WO2015144029A1 - 甲基丙烯酰基苯并咪唑酮衍生物及其抗肿瘤用途 - Google Patents

甲基丙烯酰基苯并咪唑酮衍生物及其抗肿瘤用途 Download PDF

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WO2015144029A1
WO2015144029A1 PCT/CN2015/074896 CN2015074896W WO2015144029A1 WO 2015144029 A1 WO2015144029 A1 WO 2015144029A1 CN 2015074896 W CN2015074896 W CN 2015074896W WO 2015144029 A1 WO2015144029 A1 WO 2015144029A1
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group
akt
formula
tumor
alkyl
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PCT/CN2015/074896
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English (en)
French (fr)
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李家鹏
姬志勤
周晓亚
魏少鹏
钱勇
谢天培
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上海诗丹德生物技术有限公司
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Priority to CN201580016136.4A priority Critical patent/CN106794173B/zh
Priority to US15/128,655 priority patent/US9738612B2/en
Publication of WO2015144029A1 publication Critical patent/WO2015144029A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/24—Benzimidazoles; Hydrogenated benzimidazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
    • C07D235/26—Oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention is in the field of biomedicine, and in particular, the present invention relates to antitumor use of methacryloylbenzimidazolone derivatives.
  • Tumors are the most serious public health problems facing humans. In recent years, tumors have caused millions of deaths every year in the world. The development of high-efficiency and low-toxic anti-tumor drugs has become the key to cancer treatment. At present, the focus of anti-tumor drug research and development has shifted from the development of cytotoxic drugs to the development of targeted drugs. The successful listing of imatinib has made people see the hope of curing tumors, and the development of small molecule targeted drugs has entered a period of vigorous development. Various tumor-related signal pathway inhibitors have been listed and have achieved great success. .
  • Benzimidazolone is an oxidized derivative of a 2-carbon atom on the imidazole ring and has a very wide range of uses as an intermediate in the dye industry (Jolanta S. et al. Days and pigments. 2001, 15-27).
  • benzimidazolone derivatives also have significant pharmacological activities, and are widely used as clinical drugs.
  • the second-generation antihistamine drug oxazide is a benzimidazolone derivative (Iwamoto K., et Al. Arzneistoff-aba-drug Research. 2001, 51: 971-976.).
  • a methacryloylbenzimidazolone derivative, an optical isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof for the preparation of:
  • the methacryl benzimidazolone derivative has the structure of Formula I;
  • Z 1 represents: hydroxy, C 1 -C 8 alkoxy, C 3 -C 8 alkenyloxy or C 3 -C 8 alkynyloxy, amino, or 1 or 2 C 1 -C 8 Substituted amine group;
  • Z 2 represents: hydrogen, C 1 -C 4 alkyl, C 3 -C 8 alkenyl or C 3 -C 8 alkynyl, C 1 -C 4 haloalkyl, C 1 -C 8 fatty acyl, C 1 to C 8 halo fatty acyl group, carbamoyl group, N-methylaminoformyl group, N-ethylaminoformyl group, N,N-dimethylaminoformyl group, N,N-diethyl Amino formyl, phenyl, benzoyl, phenylacetyl, benzenesulfonyl or phenyl optionally substituted by 1 to 3 substituents selected from benzoyl or phenylsulfonyl: halogen, C 1 ⁇ C 8 alkyl, C 3 -C 8 cycloalkyl, hydroxy, C 1 -C 4 alkoxy, C 1 -C 4 haloalky
  • Z 3 represents hydrogen, C 1 -C 8 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 alkenyl, C 3 -C 8 alkynyl, C 1 -C 4 haloalkyl, a C 1 -C 8 acyl group, or a 5- or 6-membered heterocyclic group having 1 to 3 hetero atoms selected from nitrogen, oxygen and sulfur, phenyl, benzoyl, phenylacetyl, phenylsulfonyl or a phenyl group optionally substituted by 1 to 3 substituents selected from the group consisting of benzoyl or benzenesulfonyl: halogen, C 1 -C 8 alkyl, C 3 -C 8 cycloalkyl, hydroxy, C 1 - C 4 alkoxy, C 1 -C 4 haloalkyl, C 1 -C 4 haloalkoxy, C 1 -C 4 haloalkyl
  • the R represents: hydrogen, ethyl, n-propyl, isopropyl, propenyl, isopropenyl, 2-cyanoethyl, benzyl, 2-fluoroethyl, 2- Chloroethyl, 2-bromoethyl, 2-methoxyethyl, 2-methoxypropyl, 2-methoxyisopropyl, 2-ethoxyethyl, 2-ethoxypropyl, 2-ethoxy
  • Y represents: hydrogen, or 1 to 4 substituents selected from the group consisting of fluorine, chlorine, bromine, nitro, methyl, ethyl, n-propyl, isopropyl, 2-chloroethyl, 2-chloropropane Base, 2-chlorobutyl, 2-bromoethyl, 2-bromopropyl, 2-bromobutyl;
  • Z 1 represents: hydroxy, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, amino, N-methylamino, N-ethylamino, N, N-di Methylamino, N,N-diethylamino;
  • Z 2 represents: hydrogen, methyl, ethyl, propyl, isopropyl, 2-chloromethyl, 2-chloroethyl, 2-chloropropyl, formyl, acetyl, phenyl, phenyl An acyl group, a benzenesulfonyl group, or a phenyl group optionally substituted by 1 to 3 substituents selected from the group consisting of benzoyl, phenylacetyl or benzenesulfonyl: fluorine, chlorine, bromine, methyl, ethyl, propyl Base, isopropyl, 2-chloroethyl, 2-chloropropyl, cyclopropane, hydroxy, methoxy, ethoxy, 2-chloroethyl, 2-chloropropyl, 2-chlorobutyl, 2 -Bromoethyl, 2-bromopropyl, 2-bromobutyl,
  • Z 3 represents hydrogen, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, propenyl, isopropenyl, propynyl, 2-chloroethyl, 2-chloropropyl, 2- Chlorobutyl, 2-bromoethyl, 2-bromopropyl, 2-bromobutyl, formyl, acetyl, n-propionyl, isopropionyl, phenyl, benzoyl, phenylacetyl, phenylsulfonyl Or a phenyl group optionally substituted by 1 to 3 substituents selected from the group consisting of benzoyl or benzenesulfonyl: halogen, C 1 -C 8 alkyl, C 3 -C 8 cycloalkyl, hydroxy, C 1 -C 4 alkoxy, C 1 -C 4 haloalkyl, C 1 -
  • the R represents: hydrogen, propenyl, isopropenyl, benzyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, 2-methoxyethyl, 2-methoxypropyl, 2-methoxyisopropyl, 2-ethoxyethyl, 2-ethoxypropyl, 2-ethoxyisopropyl;
  • Z 1 represents: hydroxy, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, amino, N-methylamino, N-ethylamino, N, N-di Methylamino, N,N-diethylamino;
  • Z 2 represents: hydrogen, formyl, acetyl, benzoyl, benzenesulfonyl, or a benzoyl optionally substituted by 1 to 3 substituents selected from the group consisting of phenylacetyl or phenylsulfonyl: Fluorine, chlorine, bromine, hydroxyl, methoxy, ethoxy, nitro, cyano;
  • Z 3 represents hydrogen, formyl, acetyl, n-propionyl, isopropionyl, benzoyl, phenylacetyl, benzenesulfonyl
  • Z 4 represents hydrogen, methyl, ethyl, n-propyl, isopropyl Base, propenyl, isopropenyl, propynyl, 2-chloroethyl, 2-chloropropyl, 2-chlorobutyl, 2-bromoethyl, 2-bromopropyl, 2-bromobutyl, or Z 3 and Z 4 together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclic group.
  • the R represents: hydrogen, propenyl, isopropenyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, 2-methoxyethyl, 2-methoxy Propyl, 2-methoxyisopropyl;
  • Z 1 represents: hydroxy, methoxy, ethoxy, n-propoxy, isopropoxy or n-butoxy.
  • the methacryl benzimidazolone derivative has the structure shown in Formula II,
  • A is selected from: C, or N;
  • R 1, R 5 are each independently selected from: hydrogen, halo, C1 ⁇ C10 alkyl group, C2 ⁇ C8 alkenyl, C2 ⁇ C8 alkynyl, CN, NO 2;
  • R 2 and R 3 are each independently selected from the group consisting of hydrogen, halogen, C1-C10 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C10 cycloalkyl, heterocyclic, aryl, heteroaryl. , C1-C10 aldehyde group, C2-C10 acyl group, C2-C10 ester group, CN, NO 2 ;
  • R 4 is selected from the group consisting of: O, S, halogen
  • alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, aldehyde, acyl, ester group is substituted or unsubstituted, and/or linear or branched of.
  • A is N.
  • R 1 is selected from the group consisting of hydrogen, halogen or trifluoromethyl.
  • R 5 is selected from the group consisting of hydrogen, halogen or trifluoromethyl.
  • R 2 and R 3 are each independently selected from substituted or unsubstituted: C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 2 -C 8 aldehyde, C 2 -C8 acyl group, C2 to C8 ester group.
  • R 4 is O.
  • the pharmaceutical composition or agent is used to reduce the level of Akt activation.
  • the pharmaceutical composition or agent is also used to inhibit phosphorylation of Akt.
  • the phosphorylation comprises phosphorylation of the Akt S473 site.
  • the methacryl benzimidazolone derivative is selected from the group consisting of compounds of the formula FD1 to FD18:
  • the methacryl benzimidazolone derivative is selected from the group consisting of compounds of the formula FD1 and FD162:
  • the tumor or tumor cell is selected from the group consisting of liver cancer, cervical cancer, glioma, colon cancer, kidney cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, skin cancer, and nasopharyngeal carcinoma. , esophageal cancer, stomach cancer, ovarian cancer and melanoma.
  • the tumor or tumor cell is a PTEN mutation (decreased PTEN expression level) or a deleted tumor or tumor cell.
  • the tumor or tumor cell is prostate cancer.
  • the tumor cells are selected from the group consisting of PC-3, A549, SGC-7901, Hela, H4, FHCC98, SMMC7721, BEL7404, A498, SW1116, MDA-MB-231, MDA-MB. - 468, DU 145, U87-MG, Wi38, H1299, or a combination thereof.
  • the tumor cells are selected from the group consisting of PC-3, A549, SGC-7901, HeLa, H4, FHCC98, SMMC7721, or a combination thereof.
  • a pharmaceutical composition comprising: (a) a methacryl benzimidazolone derivative, an optical isomer thereof, or a pharmaceutically acceptable thereof a salt or prodrug; and (b) a pharmaceutically acceptable carrier.
  • the methacryl benzimidazolone derivative comprises a compound of formula I.
  • the drug or pharmaceutical composition further comprises a cytotoxic drug (such as, but not limited to, a DNA interfering agent (such as cisplatin or doxorubicin)) and/or an antimetabolite (for example, However, it is not limited to a pyrimidine antagonist, a sputum antagonist, or a folic acid antagonist.
  • a cytotoxic drug such as, but not limited to, a DNA interfering agent (such as cisplatin or doxorubicin)
  • an antimetabolite for example, However, it is not limited to a pyrimidine antagonist, a sputum antagonist, or a folic acid antagonist.
  • the cytotoxic drug is selected from the group consisting of an anthracycline antibiotic, a paclitaxel, a vinblastine, a camptothecin, an alkylating agent, a platinum, or a combination thereof.
  • the antimetabolite is selected from the group consisting of methotrexate, pentafluorouracil, hydroxyurea, cytarabine, or a combination thereof.
  • the methacryl benzimidazolone derivative is contained in the pharmaceutical or pharmaceutical composition in an amount of from 0.01 to 99% by weight, preferably from 0.1 to 90% by weight.
  • the drug or pharmaceutical composition is also used to induce tumor cell apoptosis.
  • the drug or pharmaceutical composition is also used to interfere with tumor cell proliferation.
  • the drug or pharmaceutical composition is also used to modulate the cell cycle of tumor cells; and/or
  • the drug or pharmaceutical composition is also used to block the cell cycle.
  • the drug or pharmaceutical composition is used to induce tumor cells to produce a G1 cycle arrest.
  • the drug or pharmaceutical composition is also used to inhibit metastasis of tumor cells.
  • a method for in vitro non-therapeutic down-regulation of the activity of the PI3K/Akt pathway and/or regulation of the cell cycle comprising the steps of: in a methacryl benzimidazolone derivative, The cells are cultured in the presence of their optical isomers, or a pharmaceutically acceptable salt or prodrug thereof, thereby downregulating the activity of the PI3K/Akt pathway and/or modulating the cell cycle in the cells.
  • the cell is a tumor cell, preferably the tumor cell is selected from the group consisting of liver cancer, cervical cancer, glioma, colon cancer, kidney cancer, breast cancer, lung cancer, prostate cancer, and pancreatic cancer. , skin cancer, nasopharyngeal cancer, esophageal cancer, stomach cancer, ovarian cancer, melanoma.
  • the tumor cells are selected from the group consisting of PC-3, A549, SGC-7901, Hela, H4, FHCC98, SMMC7721, BEL7404, A498, SW1116, MDA-MB-231, MDA-MB- 468, DU145, U87-MG, Wi38, H1299 cells, or a combination thereof; more preferably selected from the group consisting of PC-3, A549, SGC-7901, HeLa, H4, FHCC98, SMMC7721 cells, or a combination thereof.
  • the compound of formula I or a pharmaceutically acceptable salt or prodrug thereof is present in an effective concentration of from 0.01 nM to 1 mM, preferably from 0.1 nM to 500 ⁇ M, most preferably from 1 nM to 100 ⁇ M (micro Moore per liter).
  • the compound of formula I has an action time of from 2 to 96 hours.
  • the compound of formula I has a duration of action of from 1 to 30 days.
  • a method for non-therapeutic inhibition of cell growth or induction of apoptosis in vitro comprising the steps of: a methacryl benzimidazolone derivative, an optical isomer thereof, The cells are cultured in the presence of a pharmaceutically acceptable salt or prodrug thereof.
  • the cell is a tumor cell.
  • the effective concentration of the methacryloylbenzimidazolone derivative is from 0.01 nM/L to 1 mM/L, preferably from 0.1 nM/L to 500 ⁇ M, most preferably 1 nM/L. -100 ⁇ M.
  • the compound of formula I has an action time of from 2 to 96 hours when used as a preparation outside a living organism.
  • a methacryloylbenzimidazolone derivative of the formula I or formula II, or a pharmaceutically acceptable salt thereof for the preparation of a composition, the composition For one or more applications selected from the group consisting of:
  • the composition is a pharmaceutical composition.
  • a sixth aspect of the invention provides a method of treating a tumor, the method comprising: administering to a subject in need of treatment a therapeutically effective amount of a compound of the formula I and / or formula II, an optical isomer thereof, or A pharmaceutically acceptable salt or prodrug, or a pharmaceutical composition thereof.
  • the administration is continued for 1 to 360 days, preferably 1 to 180 days, more preferably 1 to 60 days.
  • a pharmaceutical composition for treating or inhibiting a tumor comprising (a) a pharmaceutically acceptable carrier and (b) a compound of formula I and / or formula II An optical isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof,
  • each group is as defined above.
  • the pharmaceutical composition is in the form of an oral dosage form or an injection dosage form.
  • kits comprising:
  • a container and a methacryl benzimidazolone derivative of the formula I and/or formula II, an optical isomer thereof, or a pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier;
  • the instructions describe the use of the methacryl benzimidazolone derivative in one or more applications selected from the group consisting of:
  • the kit further includes an additional anti-cancer drug.
  • A is selected from: C, or N;
  • R 1, R 5 are each independently selected from: hydrogen, halo, C1 ⁇ C10 alkyl group, C2 ⁇ C8 alkenyl, C2 ⁇ C8 alkynyl, CN, NO 2;
  • R 2 and R 3 are each independently selected from the group consisting of hydrogen, halogen, C1-C10 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C10 cycloalkyl, heterocyclic, aryl, heteroaryl. , C1-C10 aldehyde group, C2-C10 acyl group, C2-C10 ester group, CN, NO 2 ;
  • R 4 is selected from the group consisting of: O, S, halogen
  • alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, aldehyde, acyl, ester group is substituted or unsubstituted, and/or linear or branched of.
  • A is N.
  • R 1 is selected from the group consisting of hydrogen, halogen or trifluoromethyl.
  • R 1 is selected from the group consisting of hydrogen, halogen or trifluoromethyl.
  • R 2 and R 3 are each independently selected from substituted or unsubstituted: C 1 -C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 2 -C 8 aldehyde, C 2 -C8 acyl group, C2 to C8 ester group.
  • R 4 is O.
  • the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • Figure 1 shows the effect of partial immunofluorescence assays on the phosphorylation of Akt by a portion of the FD series of compounds.
  • MK2206 (a new drug candidate developed by Merck is in the phase II clinical trial phase)
  • FD1 and FD162 can significantly reduce the phosphorylation level of Akt, and inhibit the over-activation of Akt prevalent in tumors.
  • the results show that the FD series of compounds similar to MK2206, can significantly inhibit Akt phosphorylation, thereby playing an anti-tumor effect.
  • Figure 2 shows that the compounds of the present invention are universally capable of inhibiting the proliferation of tumor cells, wherein the inhibitory activity against Hela cell proliferation is extremely high.
  • Figure 3 shows that the compound of the invention has a half-inhibitory concentration of 3.67 micromolar for Hela cell proliferation.
  • Figure 4 shows that the compounds of the invention are capable of down-regulating the PI3K/Akt/mTOR signaling pathway in a concentration dependent manner.
  • Figure 5 shows the tumor suppressing effect of the compounds of the present invention.
  • the inventors have unexpectedly discovered that methacryl benzimidazolone derivatives are effective in inhibiting the growth of tumor cells by extensive and intensive research. Further experiments have shown that this class of compounds can effectively inhibit the PI3K/Akt signaling pathway. The present invention has been completed on this basis.
  • the experimental results indicate that the methacryl benzimidazolone derivative (FD series) can significantly inhibit the phosphorylation of S473 at Akt, reduce the activation level of Akt, and thereby inhibit the activity of PI3K/Akt signaling pathway. To target anti-tumor effects. Moreover, the methacryl benzimidazolone derivative has a remarkable inhibitory effect on mTOR kinase.
  • substituted means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: the one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of: C1-C10 alkyl group, C3-C10 cycloalkyl group, C1-C10 alkoxy group, halogen, hydroxyl group, carboxyl group (-COOH), C1-C10 aldehyde group, C2-C10 acyl group, C2-C10 ester group, amino group, benzene
  • the phenyl group includes an unsubstituted phenyl group or a substituted phenyl group having 1 to 3 substituents selected from the group consisting of halogen, C1-C10 alkyl group, cyano group, OH group, nitro group, C3 group. ⁇ C10 cycloalkyl, C1-C10 alkoxy, amino.
  • C1-C10 alkyl refers to a straight or branched alkyl group having from 1 to 10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, Tert-butyl, or a similar group.
  • C3-C10 cycloalkyl refers to a cycloalkyl group having 3 to 10 carbon atoms, such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cycloheptyl group, or the like.
  • C2-C10 alkenyl refers to an alkenyl group having 1 to 10 carbon atoms, such as ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, sec-butenyl, tert-butenyl, or the like. Group.
  • C2-C10 alkynyl refers to an alkynyl group having 1 to 10 carbon atoms, such as ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, sec-butynyl, tert-butyne. Base, or a similar group.
  • C6-C10 aryl refers to an aryl group having 6 to 10 carbon atoms, including a monocyclic or bicyclic aryl group such as a phenyl group, a naphthyl group, or the like.
  • C1-C10 heteroaryl means a heteroaryl group having 1 to 10 carbon atoms, such as pyrrolyl, pyridyl, furyl, or the like.
  • C1-C10 alkoxy refers to a straight or branched alkoxy group having 1 to 10 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso Butoxy, sec-butoxy, tert-butoxy, or the like.
  • C6-C10 aryl-oxy refers to a straight or branched aryl-oxy group having 6 to 10 carbon atoms, such as phenyl-oxy, naphthyl-oxy, or the like.
  • C1-C10 heteroaryl-oxy refers to a straight or branched heteroaryl-oxy group having 1 to 10 carbon atoms, such as pyridyl-oxy, furyl-oxy, or the like. .
  • C1-C10 acyl means a group having a “-CO-alkyl” structure, preferably having a structure of "-CO-C1-C10 alkyl", such as a methyl acyl group, an ethyl acyl group, a propyl acyl group, and a different group.
  • a methyl acyl group such as a methyl acyl group, an ethyl acyl group, a propyl acyl group, and a different group.
  • C1 ⁇ C10 ester group refers to an alkyl group -COO- structure, preferably a C1 ⁇ C10 alkyl group -COO- structure, e.g. CH 3 COO-, C 2 H 5 COO-, C 3 H 8 COO-, :(CH 3 ) 2 CHCOO-, nC 4 H 9 COO-, tC 4 H 9 COO-, or the like.
  • C1 ⁇ C10 sulfonyl group refers to a "-SO 2 - alkyl” structure, preferably a "-SO 2 -C1 ⁇ C10 alkyl group” structure, e.g. methylsulfonyl, ethylsulfonyl, A propylsulfonyl group, an isopropylsulfonyl group, a butylsulfonyl group, an isobutylsulfonyl group, a sec-butylsulfonyl group, a tert-butylsulfonyl group, or the like.
  • C1-C20 heterocycloalkyl refers to a heterocycloalkyl group having 1 to 20 carbon atoms, such as an epoxyethyl group, a tetrahydrofuranyl group, a tetrahydropyrrolyl group, or the like.
  • halogen refers to F, Cl, Br and I.
  • the compound of the present invention is used interchangeably to mean a methacryloylbenzimidazolone derivative (methacryloylbenzimidazole (thio)ketone derivative), or an optical isomer thereof, a pharmaceutically acceptable salt or prodrug thereof .
  • a type of methacryloylbenzimidazolone derivative has a structure represented by the following formula I,
  • R represents: hydrogen, propenyl, isopropenyl, 2-fluoroethyl, 2-chloroethyl, 2- Bromoethyl, 2-methoxyethyl, 2-methoxypropyl, 2-methoxyisopropyl;
  • Z 1 represents: hydroxy, methoxy, ethoxy, n-propoxy, isopropoxy or n-butoxy.
  • methacryl benzimidazolone derivatives include the compounds disclosed below in Tables 1 to 4 of CN201110326737, or pharmaceutically acceptable salts thereof:
  • the compounds of the present invention also include pharmaceutically acceptable salts of methacryloylbenzimidazole (thio)ketone derivatives of the formula I.
  • pharmaceutically acceptable salt refers to a salt of a compound of the invention with a pharmaceutically acceptable inorganic and organic acid, including: hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid; Acids include: formic acid, acetic acid, propionic acid, succinic acid, naphthalene disulfonic acid (1,5), asiamic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethyl acetic acid, propylene Acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, Toluenesulfonic acid, citric acid, and amino
  • the term "pharmaceutically acceptable prodrug” refers to a compound that is inactive in vitro but is capable of being converted in vivo to an active substance of Formula I to exert its pharmacological action.
  • the invention also provides a pharmaceutical composition having significant anti-tumor efficacy comprising a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable salts Carrier.
  • the pharmaceutical composition comprises a therapeutically effective amount of anagrelide hydrochloride, and one or more pharmaceutically acceptable carriers.
  • the compound itself or a mixture of a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, diluent or the like may be administered orally in the form of a tablet, capsule, granule, powder or syrup or may be administered orally in the form of an injection. Dosing.
  • the pharmaceutical composition preferably contains, as an active ingredient, a compound of the formula I of the present invention or a pharmaceutically acceptable salt thereof in an amount of from 0.01% to 99% by weight, more preferably from 0.1% to 90% by weight of the active ingredient.
  • compositions can be prepared by conventional pharmaceutical methods.
  • useful pharmaceutical adjuvants include excipients (e.g., saccharide derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, dextrin, and carboxymethyl starch; Cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose; gum arabic; dextran; silicate derivatives such as magnesium metasilicate Aluminum; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc., binders such as gelatin, polyvinylpyrrolidone and polyethylene glycol, disintegrants For example, cellulose derivatives such as sodium carboxymethylcellulose, polyvinylpyrrolidone, lubricants (such as talc, calcium stea
  • the amount of the compound of the present invention, a pharmaceutically acceptable salt or prodrug thereof, or a pharmaceutical composition thereof, The patient's age, gender, race, condition, etc. vary.
  • the daily dose for a typical adult is from about 10 mg to about 2000 mg, preferably from 50 mg to about 1000 mg.
  • PI3K/Akt/mTOR Signaling Pathway The present invention finds that methacryloylbenzimidazolone derivatives can inhibit the PI3K/Akt signaling pathway, thereby regulating cell cycle (blocking), inhibiting tumor cell growth, and inducing apoptosis.
  • the role can be applied to the treatment of tumors and the like.
  • the results of enzyme-linked immunosorbent assay showed that the methacryl benzimidazolone derivative (FD series) can significantly inhibit the phosphorylation of S473 at Akt, reduce the activation level of Akt, and thereby inhibit the PI3K/Akt signal.
  • the activity of the pathway plays a role in targeting anti-tumor.
  • protein electrophoresis results indicate that the compound of the present invention can significantly inhibit Akt phosphorylation at the cellular level after being applied to tumor cells (e.g., A549 cells) for a period of time.
  • tumor cells e.g., A549 cells
  • the sulforhodamine B method results indicate that FD1 in the FD series compound of the present invention can significantly inhibit the proliferation of tumor cell lines and has high selectivity, and has a PTEN-deficient cell line PC-3. Better inhibition of proliferation.
  • PI3K is an important signal transduction molecule in cells. According to the structural characteristics of PI3K P110 subunit and substrate molecules, it can be divided into three categories, among which the first type of PI3K function is the most important.
  • PI3K consists mainly of a catalytic subunit P110 and a regulatory subunit P85.
  • PI3K can be activated by extracellular signal stimulation such as growth factors, cytokines, and hormones.
  • PI3K activation phosphorylates membrane phosphoinositides, catalyzing the 3 hydroxyl groups on the inositol ring to produce phosphatidylinositol-3,4-biphosphate, PI-3,4P2 and 3,4,5 Phosphadylinositol 3,4,5-trisphosphate (PI-3,4,5P3), which can act as a second messenger to transmit signals in cells, mediating various cellular functions of PI3K, such as these lipids
  • the plastid product activates Akt by binding to the PH (pleckstrin homology) region of Akt.
  • the expression product of the tumor suppressor gene pten (phosphatase and tensinhomologdelet-edonchromosometen) induces dephosphorylation of inositol 3 phosphate, thereby negatively regulating the PI3K pathway.
  • Akt is a Ser/Thr protein kinase. Under the synergistic action of phosphoinositide-dependent protein kinase (PDK), PI-3, 4P2 and PI-3, 4, 5P3 can bind to Akt, resulting in Akt translocation from cytoplasm to quality. Membrane and promote phosphorylation of Ser473 and Thr308 at Akt.
  • PDK phosphoinositide-dependent protein kinase
  • Akt activation is essential for Akt activation, and Akt activation is an important prerequisite for its ability to promote cell survival.
  • Activated Akt is mainly played by promoting phosphorylation of downstream substrates such as Bad (one of the pro-apoptotic members of the Bcl-2 family), mTOR, Caspase 3, and glycogen synthase kinase-3 (GSK-3).
  • Bad one of the pro-apoptotic members of the Bcl-2 family
  • mTOR mTOR
  • Caspase 3 glycogen synthase kinase-3
  • mTOR also known as FRAP (FKBP-rapamycin-associated protein)
  • FRAP FKBP-rapamycin-associated protein
  • PIKKs phosphoinositide kinase 3-related kinase family
  • 4EBP1 eukaryocyte promoter 4E binding protein
  • phosphorylation status of p70S6k initiate the translation process.
  • the dephosphorylated 4EBP1 binds to the translation initiation factor eIF-4E, thereby inactivating the latter, while the phosphorylated 4EBP1 dissociates and releases eIF-4E, allowing the latter to bind to the 5' end of the mRNA.
  • Translation process Phosphorylation of p70S6k initiates translation by promoting phosphorylation of the 40S ribosomal protein S6.
  • the PI3K/Akt/mTOR signaling pathway plays an important role in the proliferation and survival of normal and tumor cells. Antitumor drugs that inhibit mTOR as the main mechanism of action have entered the early stage of clinical development.
  • the invention provides a method of modulating a cell cycle agent, the method comprising administering to a subject cell an effective amount of a compound of formula I or a pharmaceutical composition comprising a compound of formula I.
  • the compounds of formula I are useful for arresting the cell cycle, such as for inducing cells to produce a G1 cycle arrest, thereby regulating the cell cycle of the cell, or altering the progression of cell division.
  • the compound of formula I or a pharmaceutical composition thereof can selectively modulate the cell cycle of a part of cells, but does not induce a cycle arrest for other non-sensitive cells.
  • the compound of formula I or a pharmaceutical composition thereof is used to modulate the cell cycle of tumor cells.
  • the tumor cells are tumor cells selected from the group consisting of Hela, H4, FHCC98, SMMC7721, BEL7404, A498, SW1116, MDA-MB-231, MDA-MB-468, DU145, U87- MG, Wi38, H1299 cells, or a combination thereof; preferably selected from the group consisting of HeLa, H4, FHCC98, SMMC7721 cells, or a combination thereof.
  • the compound of formula I can act on cells at a lower concentration, preferably at a concentration of ⁇ 1 mM/L, and induce or modulate the cell cycle of the cell, producing a periodic block.
  • the compound of formula I has an effective concentration of from 0.01 nM/L to 1 mM/L, preferably from 0.1 nM/L to 500 ⁇ M/L, most preferably from 1 nM/L to 100 ⁇ M/L.
  • the action time of the compound of the formula I is not particularly limited, and may be, for example, 2 to 30 days.
  • the duration of action of the compound of formula I differs depending on the environment of action and the subject of the action.
  • the compound of formula I has an action time of from 2 to 96 hours.
  • the compound of formula I has a duration of action of from 1 to 30 days.
  • the invention provides a method of inducing apoptosis or inhibiting cell growth, the method comprising administering to a subject cell an effective amount of a compound of formula I or a pharmaceutical composition comprising a compound of formula I.
  • the compound of formula I or a pharmaceutical composition thereof can selectively induce partial apoptosis or inhibit partial cell growth without inhibiting the growth of other non-sensitive cells.
  • the compound of formula I or a pharmaceutical composition thereof is used to modulate the cell cycle of tumor cells.
  • the tumor cells are tumor cells selected from the group consisting of Hela, H4, FHCC98, SMMC7721, BEL7404, A498, SW1116, MDA-MB-231, MDA-MB-468, DU145, U87-MG, Wi38 , H1299 cells, or a combination thereof; most preferred subject tumor cells are selected from the group consisting of HeLa, H4, FHCC98, SMMC7721 cells, or a combination thereof.
  • the compound of formula I can act on a subject cell at a lower concentration, preferably at a concentration of ⁇ 100 mM/L, and induce or modulate the cell cycle of the cell to produce a periodic block.
  • the compound of formula I has an effective concentration of from 0.01 nM/L to 1 mM/L, preferably from 0.1 nM/L to 500 ⁇ M/L, most preferably from 1 nM/L to 100 ⁇ M/L.
  • the action time of the compound of the formula I is not particularly limited, and may be, for example, 2 to 30 days.
  • the duration of action of the compound of formula I differs depending on the environment of action and the subject of the action.
  • the compound of formula I has an action time of from 2 to 96 hours.
  • the compound of formula I has a duration of action of from 1 to 30 days.
  • the invention provides a method of inducing apoptosis or inhibiting cell growth, the method comprising administering to a subject cell an effective amount of a compound of formula I or a pharmaceutical composition comprising a compound of formula I.
  • the pharmaceutical composition comprises: (a) a therapeutically effective amount of a compound of formula I; and (b) a pharmaceutically acceptable carrier.
  • the tumor comprises: cervical cancer, glioma, liver cancer.
  • the compound of formula I has an effective concentration of from 0.01 nM/L to 1 mM/L, preferably from 0.1 nM/L to 500 ⁇ M/L, most preferably from 1 nM/L to 100 ⁇ M/L.
  • the invention also provides a process for the preparation of an antitumor drug or pharmaceutical composition comprising: mixing a therapeutically effective amount of a compound of formula I with a pharmaceutically acceptable carrier to form a pharmaceutical composition.
  • the pharmaceutical composition further comprises a component selected from the group consisting of a tumor suppressor, a tumor apoptosis inducing agent, or a combination thereof.
  • Tumors can be treated or inhibited by administering to a subject a therapeutically effective amount of a compound of formula I or a pharmaceutical composition comprising a compound of formula I.
  • reagents and other materials used in the examples herein are available from commercially available sources, and biological reagents and materials are purchased from Shanghai Research Biotechnology Co., Ltd. unless otherwise specified.
  • the ELISA screening method was established by double antibody sandwich method: A549 cells (a lung cancer cell, purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were inoculated into a 12-well plate at a certain concentration, and cultured overnight in a 37 ° C carbon dioxide incubator. The cells were adhered to the wall.
  • A549 cells a lung cancer cell, purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
  • the Akt antibody was inoculated into the highly adsorbed microplate at a specific concentration and placed in a refrigerator at 4 ° C; the 12-well plate was washed three times with sterile PBS, and serum-free F12 was added, and the incubator was cultured 24 Hours; MK2206, Triciribine and FD series drugs were added to 12-well plates for 1 hour, 100 ng of IGF-1 was added to each well for 10 minutes; 12-well plates were washed three times with pre-cooled PBS, and 100 ⁇ l of RIPA lysate was added to each well.
  • the results are shown in Table 1.
  • the positive controls were MK2206 and Triciribine (tresiribine), and the inhibition rates of Akt phosphorylation at the cellular level were 92% and 46%, respectively.
  • the inhibition rates of FD1 and FD3 on Akt phosphorylation in the compounds of the present invention were respectively 85% and 91% were superior to the positive control Triciribine, which was comparable to the positive control MK2206.
  • FD2, FD4, FD12, FD13 were equivalent to Triciribine.
  • A549 cells were seeded into a 12-well plate at a density of 2 ⁇ 10 5 cells/well, adhered to the wall overnight, discarded and incubated with physiological saline for three times, added to fresh mist serum medium for 24 hours, and added to the test compound for 1 h. Then, IGF-I was added for stimulation for 10 min, cell lysate was added and lysed on ice for half an hour, and cell lysate was collected. After centrifugation at 20000 g for 4 min at 4 ° C, the supernatant was taken, and the protein was quantified by MicroBCA protein quantification kit, and the protein concentration was adjusted. Make uniform.
  • the mixture was mixed with 4 ⁇ SDS loading buffer [200 mM Tris.Cl (pH 6.8), 400 mM DTT, 8% SDS, 0.4% bromophenol blue, 40% glycerol] and boiled for 15 min. After cooling at room temperature, an equal amount of the sample was applied to a polyacrylamide gel and electrophoresed in a Tris-glycine running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) at 80-120 V for about 2 h. The protein was transferred from the gel to a nitrocellulose filter by semi-dry method.
  • 4 ⁇ SDS loading buffer 200 mM Tris.Cl (pH 6.8), 400 mM DTT, 8% SDS, 0.4% bromophenol blue, 40% glycerol
  • the transfer conditions were: transfer buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS, 20% methanol); transfer current 25 mA; transfer time 2 h.
  • transfer buffer 39 mM glycine, 48 mM Tris base, 0.037% SDS, 20% methanol
  • transfer current 25 mA
  • transfer time 2 h 2 h.
  • Ponceau S staining was used to determine the transfer and position of the protein band on the nitrocellulose filter, followed by a blocking solution containing 5% skim milk powder [5% skim milk powder, 20 mM Tris-HCl). (pH 7.2-7.4), 150 mM NaCl, 0.1% (v/v) Tween 20] were blocked at room temperature for 2 h at room temperature.
  • a specific primary antibody was added to hybridize overnight at 4 °C.
  • the 12 lanes of Akt show that the bands are dark and the same in size, indicating that the total Akt is the same in each lane; p-Akt( The color depth and band size of the 12 lanes of S473) are very different, indicating that the amount of p-Akt (S473) is different in each band; IGF can stimulate the rapid rise of Akt phosphorylation level in the signaling pathway High, in the control 1, only 100 ng / ml of IGF was added to the cell culture medium, the phosphorylation level of Akt was significantly increased, while the control solution 2 did not add IGF to the cell culture medium, and the Akt phosphorylation level was lower; MK2206 was Merck Pharmaceuticals An Akt phosphorylation inhibitor developed is currently in the clinical phase II trial and shows good therapeutic effects on a variety of tumors.
  • the band corresponding to MK2206 is significantly more pronounced than the control 1 band. Decreased, indicating that MK2206 significantly inhibited the increase of Akt phosphorylation level after IGF stimulation; FD1 and FD3 showed comparable effects to MK2206.
  • the experimental results show that the FD series compound (methacryloylbenzimidazolone derivative) has a significant inhibitory effect on the phosphorylation of the S473 site of Akt.
  • TCA trichloroacetic acid
  • Inhibition rate (%) (OD value control hole- OD value administration hole ) / OD value control hole ⁇ 100%.
  • the results are shown in Table 2.
  • the concentration of the MK2206 and FD series compounds was 10 ⁇ M.
  • the PC-3 for prostate cancer cell line was purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
  • the A549 lung cancer cell line was purchased from the Shanghai Institute of Life Sciences, Chinese Academy of Sciences, and the SGC-7901 was a gastric cancer cell line.
  • IIIB-60 in compounds IIIB-01 to IIIB-60;
  • the present invention tested the inhibitory activity of FD162 compounds against 114 tumor-associated kinases and found that FD162 (concentration 10 uM) selectively inhibits mTOR activity. This experiment was commissioned by Eurofins Pharma Discovery Services UK Limited and a report was issued. The experimental results are shown in the table below.
  • the present invention tested the proliferation inhibitory activity of the representative compound FD162 against more than 20 tumor cell lines, and the experimental method was the same as in Example 2. The results are shown in Figure 2, indicating that FD162 can generally inhibit the proliferation of tumor cells, wherein the half-inhibitory concentration of FD162 on Hela cell proliferation is 3.67 micromolar.
  • FD162 can down-regulate the PI3K/Akt/mTOR signaling pathway in a concentration-dependent manner.
  • RAD is a marketed mTOR inhibitor antitumor drug everolimus at a concentration of 10 uM
  • Rapa is rapamycin
  • MK is an Akt inhibitor antitumor drug MK2206 in clinical trials.
  • the method used in the animal activity experiment was subcutaneous transplantation of Hela cells in nude mice.
  • the tumor tissue in the vigorous growth period was cut into 1.5 mm 3 and inoculated subcutaneously in the right axilla of nude mice under aseptic conditions.
  • the nude mice were transplanted subcutaneously with a vernier caliper to measure the diameter of the transplanted tumor.
  • the animals were randomly divided into groups.
  • FD162 was divided into three groups according to 20mg/kg, 10mg/kg and 5mg/kg, 9 in each group; rapamycin was in the group of 10mg/kg; the model group was given normal saline, once a day, intraperitoneal injection, the dosing period was 12 day.
  • the diameter of the transplanted tumor was measured twice a week during the entire experiment, and the body weight of the mice was weighed.
  • the experimental results show that the compound FD162 of the present invention has the best inhibitory effect on the tumor in the middle dose group (10 mg/kg), which is superior to the positive control group, and the tumor inhibition rate in the middle dose group (10 mg/kg) after 12 days of administration. It has reached more than 50%.
  • the tumor suppressing effect is shown in Fig. 5.
  • the FD162 compounds disclosed in the present invention can be prepared by the following synthetic route.
  • the methacryloylbenzimidazolone derivative of the present invention can down-regulate the Akt phosphorylation level in the P13K/Akt signaling pathway, and the effect is comparable to the novel small molecule targeting drug MK2206; and the cell level study finds that FD1 is The representative methacryl benzimidazolone derivatives have a good proliferation inhibitory effect on tumor cells. Unlike MK2206, FD1 has a better effect on PTEN-deficient cells.
  • the methacryloylbenzimidazolone derivative of the present invention has a remarkable inhibitory effect on mTOR kinase and can be developed as an mTOR kinase inhibitor for tumor therapy.

Abstract

本发明公开了一种甲基丙烯酰基苯并咪唑酮衍生物，其光学异构体，或其药学上可接受的盐或前药的用途，用于制备(a)用于下调PI3K/Akt通路的活性的药物组合物或试剂；(b)用于治疗或抑制肿瘤；和/或，抑制肿瘤细胞生长的药物组合物或试剂；和/或(c)用于阻滞细胞周期的药物组合物或试剂。本发明所涉及的化合物能够下调P13K/Akt信号通路中Akt磷酸化水平，作用效果与新型小分子靶向药物MK2206相当；而细胞水平的研究发现，以FD1为代表的甲基丙烯酰基苯并咪唑酮衍生物对肿瘤细胞具有良好的增殖抑制效果，与MK2206有所不同的是，FD1对PTEN缺失型的细胞作用效果更好。

Description

甲基丙烯酰基苯并咪唑酮衍生物及其抗肿瘤用途 技术领域
本发明属于生物医药领域，具体地说，本发明涉及甲基丙烯酰基苯并咪唑酮衍生物的抗肿瘤用途。
背景技术
肿瘤是目前人类所面临的最严重的公共健康问题，近年来肿瘤每年造成全球数百万人死亡，开发高效低毒的抗肿瘤药物成为肿瘤治疗的关键所在。目前，抗肿瘤药物研发的重点已经由细胞毒类药物的开发转移到靶向药物的开发。伊马替尼的成功上市，让人们看到了治愈肿瘤的希望，由此小分子靶向药物的开发进入了蓬勃发展的时期，各个与肿瘤相关的信号通路抑制剂相继上市，取得了巨大的成功。
研究发现，PI3K/Akt信号通路异常活化存在于多种肿瘤中，其中Akt磷酸化而活化是该条通路活化的关键步骤，Akt的T308位点和S473位点同时活化是Akt活化的必要条件。PI3K/Akt信号通路抑制剂开发已经成为抗肿瘤药物研发的热点，目前已有数十个该类型药物处于临床试验阶段，MK2206和Triciribine就该类型药物的典型，临床治疗结果显示效果显著，有着很好的开发前景。鉴于PI3K/Akt信号通路的异常活化在肿瘤中的普遍存在，成功开发此类药物必然能取得良好的社会效益和经济效益。
苯并咪唑酮是咪唑环上2位碳原子的氧化衍生物，在染料工业中作为中间体具有非常广泛的用途(Jolanta S.et al.Dyes and pigments.2001，15-27)。除此之外，苯并咪唑酮衍生物也具有明显的药理活性，作为临床用药普遍应用，如第二代抗组胺药物奥沙米特就属于苯并咪唑酮衍生物(Iwamoto K.，et al.Arzneimittel-forschung-drug Research.2001，51：971-976.)。中国专利申请CN201110326737.9中，申请人在在苯并咪唑酮N3位引入甲基丙烯酰基后，所得甲基丙烯酰基苯并咪唑酮(甲基丙烯酰基苯并咪唑(硫)酮)衍生物表现出了极高的抗细菌活性。同时在该专利申请中公开了此类化合物的制备方法，并给了具体结构。
发明内容
本发明的目的在于提供一种甲基丙烯酰基苯并咪唑酮衍生物在抗肿瘤治疗方面的应用。
本发明的第一方面，提供了一种甲基丙烯酰基苯并咪唑酮衍生物，其光学异构体，或其药学上可接受的盐或前药的用途，用于制备：
(a)用于下调PI3K/Akt通路的活性的药物组合物或试剂；
(b)用于治疗或抑制肿瘤和/或抑制肿瘤细胞生长的药物组合物或试剂；
(c)用于阻滞细胞周期的药物组合物或试剂；和/或
(d)用于制备抑制mTOR激酶活性的药物组合物或试剂。
在另一优选例中，所述甲基丙烯酰基苯并咪唑酮衍生物具有式I结构；
Figure PCTCN2015074896-appb-000001
其中，
R代表：
氢；C1～C8的烷基；或任选被氰基，卤素，苯环，C1～C4烷氧基或C1～C4烷硫基取代的C1～C8烷基，C3～C8链烯基或C3～C8炔基；或任选被氰基，卤素，C1～C4烷氧基或C1～C4烷硫基取代的C3～C8链烯基或C3～C8炔基；苯基或由选自下列的1～3个取代基任选取代的苯基：卤素，C1～C8烷基，C3～C8环烷基，羟基，C1～C4烷氧基，C1～C4卤代烷基，C1～C4卤代烷氧基，C1～C4卤代烷硫基，C1～C4卤代烷基磺酰基，羧基，硝基，氰基，苯基，苯氧基，苯甲酰基；
Y代表：
氢；或选自下列的1～4个取代基：卤素，硝基，C1～C8烷基，C3～C8环烷基，C1～C4卤代烷基；
其中，当Y代表氢时，R不为异丙烯基；
或者Y代表：
Figure PCTCN2015074896-appb-000002
其中，Z1代表：羟基，C1～C8烷氧基，C3～C8烯氧基或C3～C8炔氧基，氨基，或者被1个或2个C1～C8烷基取代的胺基；
或者Y代表：
-O(S)-Z2
其中，Z2代表：氢，C1～C4烷基，C3～C8链烯基或C3～C8炔基，C1～C4卤代烷基，C1～C8脂肪酰基，C1～C8卤代脂肪酰基，氨基甲酰基，N-甲基胺基甲酰基，N-乙基胺基甲酰基，N,N-二甲基胺基甲酰基，N,N-二乙基胺基甲酰基，苯基，苯甲酰基，苯乙酰基，苯磺酰基或由选自下列的1～3个取代基任选取代的苯基，苯甲酰基或苯磺酰基：卤素，C1～C8烷基，C3～C8环烷基，羟基，C1～C4烷氧基，C1～C4卤代烷基，C1～C4卤代烷氧基，C1～C4卤代烷硫基，C1～C4卤代烷基磺酰基，羧基，硝基，氰基；
或者Y代表：
Figure PCTCN2015074896-appb-000003
其中，Z3代表氢，C1～C8烷基，C3～C8环烷基，C3～C8链烯基，C3～C8链炔基，C1～C4卤代烷基，C1～C8酰基，或带有1～3个选自氮、氧和硫的杂原子的的5元或6元杂环基，苯基，苯甲酰基，苯乙酰基，苯磺酰基或由选自下列的1～3个取代基任选取代的苯基，苯甲酰基或苯磺酰基：卤素，C1～C8烷基，C3～C8环烷基，羟基，C1～C4烷氧基，C1～C4卤代烷基，C1～C4卤代烷氧基，C1～C4卤代烷硫基，C1～C4卤代烷基磺酰基，羧基，硝基，氰基；Z4代表氢，C1～C8烷基，C3～C8环烷基，C3～C8链烯基，C3～C8链炔基，C1～C4卤代烷基，或Z3和Z4与它们所连接的氮原子一起形成5～7元杂环基，除了氮原子以外，该杂环基 可以含有一个或两个以上选自氮和氧的杂原子。
在另一优选例中，所述R代表：氢，乙基，正丙基，异丙基，丙烯基，异丙烯基，2-氰乙基，苯甲基，2-氟乙基，2-氯乙基，2-溴乙基，2-甲氧乙基，2-甲氧丙基，2-甲氧异丙基，2-乙氧乙基，2-乙氧丙基，2-乙氧异丙基，苯基，或由选自下列的1～3个取代基任选取代的苯基：氟，氯，溴，甲基，乙基，正丙基，异丙基，环丙烷基，羟基，甲氧基，乙氧基，丙氧基，异丙氧基，2-氯乙基，2-氯丙基，2-氯乙氧基，2-氯丙氧基，羧基，硝基，氰基；
Y代表：氢，或选自下列的1～4个取代基：氟，氯，溴，硝基，甲基，乙基，正丙基，异丙基，2-氯乙基，2-氯丙基，2-氯丁基，2-溴乙基，2-溴丙基，2-溴丁基；
其中，当Y代表氢时，R不为异丙烯基；
或者Y代表：
Figure PCTCN2015074896-appb-000004
其中，Z1代表：羟基，甲氧基，乙氧基，正丙氧基，异丙氧基，正丁氧基，氨基，N-甲胺基，N-乙胺基，N,N-二甲胺基，N,N-二乙胺基；
或者Y代表：
-O(S)-Z2
其中，Z2代表：氢，甲基，乙基，丙基，异丙基，2-氯甲基，2-氯乙基，2-氯丙基，甲酰基，乙酰基，苯基，苯甲酰基，苯磺酰基，或由选自下列的1～3个取代基任选取代的苯基，苯甲酰基，苯乙酰基或苯磺酰基：氟，氯，溴，甲基，乙基，丙基，异丙基，2-氯乙基，2-氯丙基，环丙烷，羟基，甲氧基，乙氧基，2-氯乙基，2-氯丙基，2-氯丁基，2-溴乙基，2-溴丙基，2-溴丁基，氯甲氧基，2-氯乙氧基，2-氯丙氧基，溴甲氧基，2-溴乙氧基，2-溴丙氧基，羧基，硝基，氰基；
或者，Y代表：
Figure PCTCN2015074896-appb-000005
其中，Z3代表氢，甲基，乙基，正丙基，异丙基，环丙烷基，丙烯基，异丙烯基，丙炔基，2-氯乙基，2-氯丙基，2-氯丁基，2-溴乙基，2-溴丙基，2-溴丁基，甲酰基，乙酰基，正丙酰基，异丙酰基，苯基，苯甲酰基，苯乙酰基，苯磺酰基或由选自下列的1～3个取代基任选取代的苯基，苯甲酰基或苯磺酰基：卤素，C1～C8烷基，C3～C8环烷基，羟基，C1～C4烷氧基，C1～C4卤代烷基，C1～C4卤代烷氧基，C1～C4卤代烷硫基，C1～C4卤代烷基磺酰基，羧基，硝基，氰基；Z4代表氢，甲基，乙基，正丙基，异丙基，丙烯基，异丙烯基，丙炔基，2-氯乙基，2-氯丙基，2-氯丁基，2-溴乙基，2-溴丙基，2-溴丁基，或Z3和Z4与它们所连接的氮原子一起形成5～7元杂环基。
在另一优选例中，所述R代表：氢，丙烯基，异丙烯基，苯甲基，2-氟乙基，2-氯乙基，2-溴乙基，2-甲氧乙基，2-甲氧丙基，2-甲氧异丙基，2-乙氧乙基，2-乙氧丙基，2-乙氧异丙基；
Y代表：
Figure PCTCN2015074896-appb-000006
其中，Z1代表：羟基，甲氧基，乙氧基，正丙氧基，异丙氧基，正丁氧基，氨基，N-甲胺基，N-乙胺基，N,N-二甲胺基，N,N-二乙胺基；
或者Y代表：
-O(S)-Z2
其中，Z2代表：氢，甲酰基，乙酰基，苯甲酰基，苯磺酰基，或由选自下列的1～3个取代基任选取代的苯甲酰基，苯乙酰基或苯磺酰基：氟，氯，溴，羟基，甲氧基，乙氧基，硝基，氰基；
或者，Y代表：
Figure PCTCN2015074896-appb-000007
其中，Z3代表氢，甲酰基，乙酰基，正丙酰基，异丙酰基，苯甲酰基，苯乙酰基，苯磺酰基；Z4代表氢，甲基，乙基，正丙基，异丙基，丙烯基，异丙烯基，丙炔基，2-氯乙基，2-氯丙基，2-氯丁基，2-溴乙基，2-溴丙基，2-溴丁基，或Z3和Z4与它们所连接的氮原子一起形成5～7元杂环基。
在另一优选例中，所述R代表：氢，丙烯基，异丙烯基，2-氟乙基，2-氯乙基，2-溴乙基，2-甲氧乙基，2-甲氧丙基，2-甲氧异丙基；
Y代表：
Figure PCTCN2015074896-appb-000008
其中，Z1代表：羟基，甲氧基，乙氧基，正丙氧基，异丙氧基，正丁氧基。
在另一优选例中，所述甲基丙烯酰基苯并咪唑酮衍生物具有式II所示结构，
Figure PCTCN2015074896-appb-000009
式II中，
A选自：C、或N；
R1、R5分别独立地选自：氢、卤素、C1～C10烷基、C2～C8烯基、C2～C8炔基、CN、NO2；
R2、R3分别独立地选自：氢、卤素、C1～C10烷基、C2～C8烯基、C2～C8炔基、C3～C10环烷基、杂环基、芳基、杂芳基、C1～C10醛基、C2～C10酰基、C2～C10酯基、CN、NO2；
R4选自：O、S、卤素；
Figure PCTCN2015074896-appb-000010
表示单键或双键；
所述烷基、烯基、炔基、环烷基、杂环基、芳基、杂芳基、醛基、酰基、酯基为取代的或非取代的，和/或直链的或支链的。
在另一优选例中，A为N。
在另一优选例中，R1选自：氢、卤素或三氟甲基。
在另一优选例中，R5选自：氢、卤素或三氟甲基。
在另一优选例中，R2、R3分别独立地选自，取代的或未取代的：C1～C8烷基、C2～C8烯基、C2～C8炔基、C2～C8醛基、C2～C8酰基、C2～C8酯基。
在另一优选例中，R4为O。
在另一优选例中，所述的药物组合物或试剂用于降低Akt活化水平。
在另一优选例中，所述的药物组合物或试剂还用于抑制Akt的磷酸化。
在另一优选例中，所述磷酸化包括Akt S473位点的磷酸化。
在另一优选例中，所述的甲基丙烯酰基苯并咪唑酮衍生物选自式FD1至FD18所示结构的化合物：
Figure PCTCN2015074896-appb-000011
Figure PCTCN2015074896-appb-000012
或其组合。
在另一优选例中，所述甲基丙烯酰基苯并咪唑酮衍生物选自式FD1和FD162所示结构的化合物：
Figure PCTCN2015074896-appb-000013
或其组合。
在另一优选例中，所述的肿瘤或肿瘤细胞选自：肝癌、宫颈癌、神经胶质瘤、结肠癌、肾癌、乳腺癌、肺癌、前列腺癌、胰腺癌、皮肤癌、鼻咽癌、食道癌、胃癌、卵巢癌和黑色素瘤。
在另一优选例中，所述肿瘤或肿瘤细胞为PTEN突变(PTEN表达水平下降)或缺失的肿瘤或肿瘤细胞。
在另一优选例中，所述肿瘤或肿瘤细胞为前列腺癌。
在另一优选例中，所述的肿瘤细胞选自下组：PC-3、A549、SGC-7901、Hela、H4、FHCC98、SMMC7721、BEL7404、A498、SW1116、MDA-MB-231、MDA-MB-468、DU145、U87-MG、Wi38、H1299，或其组合。
在另一优选例中，所述肿瘤细胞选自下组：PC-3、A549、SGC-7901、Hela、H4、FHCC98、SMMC7721、或其组合。
本发明的第二方面，提供了一种药物组合物，所述的药物组合物包括：(a)甲基丙烯酰基苯并咪唑酮衍生物，其光学异构体，或其药学上可接受的盐或前药；和(b)药学上可接受的载体。
在另一优选例，所述的甲基丙烯酰基苯并咪唑酮衍生物包括式I化合物。
在另一优选例中，所述药物或药物组合物还包括细胞毒类药物(例如，但不限于DNA干扰剂(例如顺铂或多柔比星))和/或抗代谢类药物(例如，但不限于嘧啶拮抗物、嘌呤拮抗物、叶酸拮抗物)。
在另一优选例中，所述细胞毒类药物选自：蒽环类抗生素、紫杉醇类、长春碱类、喜树碱类、烷化剂类、铂类或其组合。
在另一优选例中，所述抗代谢类药物选自：甲氨蝶呤、五氟尿嘧啶、羟基脲、阿糖胞苷或其组合。
在另一优选例中，所述药物或药物组合物中甲基丙烯酰基苯并咪唑酮衍生物的含量为0.01-99wt％，较佳地为0.1-90wt％。
在另一优选例中，所述药物或药物组合物还用于诱导肿瘤细胞凋亡。
在另一优选例中，所述药物或药物组合物还用于干扰肿瘤细胞增殖。
在另一优选例中，所述药物或药物组合物还用于调控肿瘤细胞的细胞周期；和/或
所述药物或药物组合物还用于阻滞细胞周期。
在另一优选例中，所述药物或药物组合物用于诱导肿瘤细胞产生G1周期阻滞。
在另一优选例中，所述药物或药物组合物还用于抑制肿瘤细胞的转移。
本发明的第三方面，提供了一种体外非治疗性的下调PI3K/Akt通路的活性和/或调节细胞周期的方法，所述方法包括步骤：在甲基丙烯酰基苯并咪唑酮衍生物，其光学异构体，或其药学上可接受的盐或前药存在下，培养所述细胞，从而在所述细胞中下调PI3K/Akt通路的活性和/或调节细胞周期。
在另一优选例中，所述细胞为肿瘤细胞，较佳地所述肿瘤细胞选自：肝癌、宫颈癌、神经胶质瘤、结肠癌、肾癌、乳腺癌、肺癌、前列腺癌、胰腺癌、皮肤癌、鼻咽癌、食道癌、胃癌、卵巢癌、黑色素瘤。
在另一优选例中，所述肿瘤细胞选自下组：PC-3、A549、SGC-7901、Hela、H4、FHCC98、SMMC7721、BEL7404、A498、SW1116、MDA-MB-231、MDA-MB-468、DU145、U87-MG、Wi38、H1299细胞，或其组合；更佳地选自下组：PC-3、A549、SGC-7901、Hela、H4、FHCC98、SMMC7721细胞，或其组合。
在另一优选例中，所述式I化合物或其药学上可接受的盐或前药的有效浓度为0.01nM-1mM，较佳地为0.1nM-500μM，最佳地为1nM-100μM(微摩尔每升)。
在另一优选例中，所述式I化合物的作用时间为2～96h。
在另一优选例中，所述式I化合物的作用时间为1～30天。
本发明的第四方面，提供了一种体外非治疗性抑制细胞生长或诱导细胞凋亡的方法，所述方法包括步骤：在甲基丙烯酰基苯并咪唑酮衍生物，其光学异构体， 或其药学上可接受的盐或前药存在下，培养所述细胞。
在另一优选例中，所述细胞为肿瘤细胞。
在另一优选例中，所述甲基丙烯酰基苯并咪唑酮衍生物的有效浓度为0.01nM/L-1mM/L，较佳地为0.1nM/L-500μM，最佳地为1nM/L-100μM。
在另一优选例中，当作用于生物体体外时，所述式I化合物的作用时间为2～96h。
本发明的第五方面，提供了一种式I或式II所示的甲基丙烯酰基苯并咪唑酮衍生物或其药学上可接受的盐的用途，用于制备组合物，所述组合物用于选自下组的一种或多种应用：
(i)抑制肿瘤细胞生长；
(ii)诱导肿瘤细胞凋亡；
(iii)干扰肿瘤细胞增殖；
(iv)调控肿瘤细胞的细胞周期；
(v)抑制肿瘤细胞的转移；
(vi)下调PI3K/Akt通路的活性；
(vii)降低Akt活化水平；
(viii)抑制Akt的磷酸化；
(ix)抑制Akt S473位点的磷酸化；
(x)治疗或预防肿瘤；
(xi)降低mTOR活化水平；
(xii)抑制mTOR的磷酸化；和
(xii)下调PI3K/Akt/mTOR信号通路的活性。
在另一优选例中，所述的组合物为药物组合物。
本发明的第六方面，提供了一种治疗肿瘤的方法，所述方法包括：对需要治疗的对象施用治疗有效量的所述式I和/或式II化合物，其光学异构体，或其药学上可接受的盐或前药，或其药物组合物。
在另一优选例中，所述施用持续1～360天，较佳地1～180天，更佳地为1～60天。
本发明的第七方面，提供了一种用于治疗或抑制肿瘤的药物组合物，所述的药物组合物含有(a)药学上可接受的载体和(b)式I和/或式II化合物，其光学异构体，或其药学上可接受的盐或前药，
Figure PCTCN2015074896-appb-000014
式中，各基团的定义如上所述。
在另一优选例中，所述的药物组合物的剂型为口服剂型、注射剂型。
本发明的第八方面，提供了一种药盒，所述药盒包含：
一容器，以及位于所述容器内的式I和/或式II所示的甲基丙烯酰基苯并咪唑酮衍生物，其光学异构体，或其药学上可接受的盐或前药，以及药学上可接受的载体；
一说明书，所述说明书记载了将所述甲基丙烯酰基苯并咪唑酮衍生物用于选自下组的一种或多种应用：
(i)抑制肿瘤细胞生长；
(ii)诱导肿瘤细胞凋亡；
(iii)干扰肿瘤细胞增殖；
(iv)调控肿瘤细胞的细胞周期；
(v)抑制肿瘤细胞的转移；
(vi)下调PI3K/Akt通路的活性；
(vii)降低Akt活化水平；
(viii)抑制Akt的磷酸化；
(ix)抑制Akt S473位点的磷酸化；
(x)治疗或预防肿瘤；
(xi)降低mTOR活化水平；
(xii)抑制mTOR的磷酸化；和
(xii)下调PI3K/Akt/mTOR信号通路的活性。
在另一优选例中，所述药盒还包括额外的抗癌药物。
本发明的第九方面，提供了一种化合物，所述化合物具有式II所示结构，
Figure PCTCN2015074896-appb-000015
式II中，
A选自：C、或N；
R1、R5分别独立地选自：氢、卤素、C1～C10烷基、C2～C8烯基、C2～C8炔基、CN、NO2；
R2、R3分别独立地选自：氢、卤素、C1～C10烷基、C2～C8烯基、C2～C8炔基、C3～C10环烷基、杂环基、芳基、杂芳基、C1～C10醛基、C2～C10酰基、C2～C10酯基、CN、NO2；
R4选自：O、S、卤素；
Figure PCTCN2015074896-appb-000016
表示单键或双键；
所述烷基、烯基、炔基、环烷基、杂环基、芳基、杂芳基、醛基、酰基、酯基为取代的或非取代的，和/或直链的或支链的。
在另一优选例中，A为N。
在另一优选例中，R1选自：氢、卤素或三氟甲基。
在另一优选例中，R1选自：氢、卤素或三氟甲基。
在另一优选例中，R2、R3分别独立地选自，取代的或未取代的：C1～C8烷基、C2～C8烯基、C2～C8炔基、C2～C8醛基、C2～C8酰基、C2～C8酯基。
在另一优选例中，R4为O。
在另一优选例中，所述化合物为
Figure PCTCN2015074896-appb-000017
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示蛋白质免疫印迹测定部分FD系列化合物对Akt磷酸化的影响。
如图所示，MK2206(默克公司开发的候选新药，处于临床二期试验阶段)，FD1，FD162都能显著的降低Akt磷酸化水平，抑制肿瘤中普遍存在的Akt过度活化是肿瘤药物开发的重要策略，本结果表明FD系列化合物与MK2206作用类似，都能显著抑制Akt磷酸化，从而起到抗肿瘤作用。
图2显示了本发明化合物能够普遍的抑制肿瘤细胞的增殖，其中对Hela细胞增殖的抑制活性极高。
图3显示了本发明化合物对Hela细胞增殖的半数抑制浓度为3.67微摩尔。
图4显示了本发明化合物能够浓度依赖的下调PI3K/Akt/mTOR信号通路。
图5显示了本发明化合物的肿瘤抑制效果。
具体实施方式
本发明人通过广泛而深入的研究，意外地发现甲基丙烯酰基苯并咪唑酮衍生物可有效地抑制肿瘤细胞的生长。进一步实验证明，该类化合物能够有效抑制PI3K/Akt信号通路。在此基础上完成了本发明。
具体地，实验结果表明，所述甲基丙烯酰基苯并咪唑酮衍生物(FD系列)能够显著抑制Akt的S473位点磷酸化，降低Akt活化水平，从而抑制PI3K/Akt信号通路的活性，起到靶向抗肿瘤的作用。而且甲基丙烯酰基苯并咪唑酮衍生物对mTOR激酶具有及其显著的抑制效果。
术语
如本文所用，术语“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：指基团上的一个或多个氢原子被选自下组的取代基取代：C1～C10烷基、C3～C10环烷基、C1～C10烷氧基、卤素、羟基、羧基(-COOH)、C1～C10醛基、C2～C10酰基、C2～C10酯基、氨基、苯基；所述的苯基包括未取代的苯基或具有1-3个取代基的取代苯基，所述取代基选自：卤素、C1-C10烷基、氰基、OH、硝基、C3～C10环烷基、C1～C10烷氧基、氨基。
术语“C1～C10烷基”指具有1～10个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。
术语“C3～C10环烷基”指具有3～10个碳原子的环烷基，例如环丙基、环丁基、环戊基、环庚基、或类似基团。
术语“C2～C10烯基”指具有1～10个碳原子的烯基，例如乙烯基、丙烯基、异丙烯基、丁烯基、异丁烯基、仲丁烯基、叔丁烯基、或类似基团。
术语“C2～C10炔基”指具有1～10个碳原子的炔基，例如乙炔基、丙炔基、异丙炔基、丁炔基、异丁炔基、仲丁炔基、叔丁炔基、或类似基团。
术语“C6～C10芳基”指具有6～10个碳原子的芳基，包括单环或二环芳基，例如苯基、萘基，或类似基团。
术语“C1～C10杂芳基”指具有1～10个碳原子的杂芳基，例如吡咯基、吡啶基、呋喃基，或类似基团。
术语“C1～C10烷氧基”指具有1-10个碳原子的直链或支链烷氧基，例如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、仲丁氧基、叔丁氧基、或类似基团。
术语“C6～C10芳基-氧基”指具有6-10个碳原子的直链或支链芳基-氧基，例如苯基-氧基、萘基-氧基，或类似基团。
术语“C1～C10杂芳基-氧基”指具有1-10个碳原子的直链或支链杂芳基-氧基，例如吡啶基-氧基、呋喃基-氧基，或类似基团。
术语“C1～C10酰基”指具有“-CO-烷基”结构，优选为具有“-CO-C1～C10烷基”结构的基团，例如甲基酰基、乙基酰基、丙基酰基、异丙基酰基、丁基酰基、异丁基酰基、仲丁基酰基、叔丁基酰基、或类似基团。
术语“C1～C10酯基”指具有烷基-COO-结构，优选为具有C1～C10烷基-COO-结构的基团，例如CH3COO-、C2H5COO-、C3H8COO-、：(CH3)2CHCOO-、nC4H9COO-、tC4H9COO-，或类似基团。
术语“C1～C10磺酰基”指具有“-SO2-烷基”结构，优选为具有“-SO2-C1～C10烷基”结构的基团，例如甲基磺酰基、乙基磺酰基、丙基磺酰基、异丙基磺酰基、丁基磺酰基、异丁基磺酰基、仲丁基磺酰基、叔丁基磺酰基、或类似基团。
术语“C1～C20杂环烷基”指具有1～20个碳原子的杂环烷基，如环氧乙基、四氢呋喃基、四氢吡咯基，或类似基团。
术语“卤素”指F、Cl、Br和I。
活性成分
在本发明中，“本发明化合物”、“本发明FD系列化合物”和“本发明活性成 分”可互换使用，指甲基丙烯酰基苯并咪唑酮衍生物(甲基丙烯酰基苯并咪唑(硫)酮衍生物)，或其光学异构体，药学上可接受的盐或前药。
本发明中，一类甲基丙烯酰基苯并咪唑酮衍生物具有如下通式I所示结构，
Figure PCTCN2015074896-appb-000018
式I中R、Y的定义如上所述。
此外，结构式中“＝O(S)”表示“＝O”(酮)或“＝S”(硫酮)。
在本发明中，一类特别优选甲基丙烯酰基苯并咪唑(硫)酮衍生物中，R代表：氢，丙烯基，异丙烯基，2-氟乙基，2-氯乙基，2-溴乙基，2-甲氧乙基，2-甲氧丙基，2-甲氧异丙基；
Y代表：
Figure PCTCN2015074896-appb-000019
其中，Z1代表：羟基，甲氧基，乙氧基，正丙氧基，异丙氧基，正丁氧基。
代表性的甲基丙烯酰基苯并咪唑酮衍生物包括公开于CN201110326737中表1-表4中以下的化合物，或其药学上可接受的盐：
(1)化合物IA-01至IA-195；
(2)化合物Ia-01至Ia-195；
(3)化合物IB-01至IB-195；
(4)化合物Ib-01至Ib-195；
(5)化合物IC-01至IC-195；
(6)化合物Ic-01至Ic-195；
(7)化合物ID-01至ID-195；
(8)化合物Id-01至Id-195；
(9)化合物IIA-01至IIA-45；
(10)化合物IIa-01至IIa-45；
(11)化合物IIB-01至IIB-45；
(12)化合物IIb-01至IIb-45；
(13)化合物IIC-01至IIC-45；
(14)化合物IIc-01至IIc-45；
(15)化合物IID-01至IID-45；
(16)化合物IId-01至IId-45；
(17)化合物IIIA-01至IIIA-60；
(18)化合物IIIa-01至IIIa-60；
(19)化合物IIIB-01至IIIB-60；
(20)化合物IIIb-01至IIIb-60；
(21)化合物IIIC-01至IIIC-60；
(22)化合物IIIc-01至IIIc-60；
(23)化合物IIID-01至IIID-60；
(24)化合物IIId-01至IIId-60；
(25)化合物IVA-01至IVA-51；
(26)化合物IVa-01至IVa-51；
(27)化合物IVB-01至IVB-51；
(28)化合物IVb-01至IVb-51；
(29)化合物IVC-01至IVC-51；
(30)化合物IVc-01至IVc-51；
(31)化合物IVD-01至IVD-51；
(32)化合物IVd-01至IVd-51。
药学上可接受的盐或前药
本发明涉及的化合物还包括如通式I所示的甲基丙烯酰基苯并咪唑(硫)酮衍生物药学上可以接受的盐。
如本文所用，术语“药学上可接受的盐”指本发明化合物与药学上可接受的无机酸和有机酸所形成的盐，无机酸包括：盐酸、氢溴酸、磷酸、硝酸、硫酸；有机酸包括：甲酸、乙酸、丙酸、丁二酸、萘二磺酸(1,5)、亚细亚酸、草酸、酒石酸、乳酸、水杨酸、苯甲酸、戊酸、二乙基乙酸、丙二酸、琥珀酸、富马酸、庚二酸、己二酸、马来酸、苹果酸、氨基磺酸、苯丙酸、葡糖酸、抗坏血酸、烟酸、异烟酸、甲磺酸、对甲苯磺酸、柠檬酸，以及氨基酸。术语“药学上可接受的盐”也可以指本发明化合物的钠盐、钾盐或铵盐
如本文所用，术语“药学上可接受的前药”指在体外无活性，但能够在生物体内转化为式I所示活性物质，从而发挥其药理作用的化合物。
药物组合物
本发明还提供了一种药物组合物，其具有显著的抗肿瘤功效，其中含有治疗有效量的所述式I化合物或其药学上可接受的盐，以及一种或多种药学上可接受的载体。在本发明的另一优选例中，所述的药物组合物中含有治疗有效量的阿那格雷盐酸盐，以及一种或多种药学上可接受的载体。
可将化合物本身或其药学上可接受的盐与可药用赋形剂、稀释剂等的混合物以片剂、胶囊、颗粒剂、散剂或糖浆剂的形式口服给药或以注射剂的形式非口服给药。该药物组合物优选含有重量比为0.01％-99％的本发明的式I化合物或其药学上可接受的盐作为活性成分，更优选含有重量比为0.1％-90％的活性成分。
上述制剂可通过常规制药方法制备。可用的药用辅剂的例子包括赋形剂(例如糖类衍生物如乳糖、蔗糖、葡萄糖、甘露糖醇和山梨糖醇；淀粉衍生物如玉米淀粉、土豆淀粉、糊精和羧甲基淀粉；纤维素衍生物如结晶纤维素、羟丙基纤维素、羧甲基纤维素、羧甲基纤维素钙、羧甲基纤维素钠；阿拉伯胶；右旋糖酐；硅酸盐衍生物如偏硅酸镁铝；磷酸盐衍生物如磷酸钙；碳酸盐衍生物如碳酸钙；硫酸盐衍生物如硫酸钙等)、粘合剂(例如明胶、聚乙烯吡咯烷酮和聚乙二醇)、崩解剂(例如纤维素衍生物如羧甲基纤维素钠、聚乙烯吡咯烷酮)、润滑剂(例如滑石、硬脂酸钙、硬脂酸镁、鲸蜡、硼酸、苯甲酸钠、亮氨酸)、稳定剂(对羟基苯甲酸甲酯、对羟基苯甲酸丙酯等)、矫味剂(例如常用的甜味剂、酸味剂和香料等)、稀释剂和注射液用溶剂(例如水、乙醇和甘油等)。
本发明的化合物、其药学上可接受的盐或前药，或其药物组合物的给药量随 患者的年龄、性别、种族、病情等的不同而不同。一般成人的日给药量为大约10mg-2000mg，优选50mg-1000mg。
PI3K/Akt/mTOR信号通路本发明发现甲基丙烯酰基苯并咪唑酮衍生物能够抑制PI3K/Akt信号通路，因而具有调控细胞周期(阻滞作用)，抑制肿瘤细胞生长，诱导细胞凋亡等多种作用，可应用于肿瘤的治疗等方面。
在本发明一个实例中，酶联免疫吸附结果表明，甲基丙烯酰基苯并咪唑酮衍生物(FD系列)能够显著抑制Akt的S473位点磷酸化，降低Akt活化水平，从而抑制PI3K/Akt信号通路的活性，起到靶向抗肿瘤的作用。
在本发明一个实例中，蛋白质电泳结果表明，本发明化合物作用于肿瘤细胞(如A549细胞)一段时间后，能够在细胞水平显著抑制Akt的磷酸化。
在本发明一个实例中，磺酰罗丹明B法结果表明，本发明FD系列化合物中的FD1能够显著抑制肿瘤细胞株增殖，并且具有很高的选择性，对PTEN缺失的细胞株PC-3具有更好的增殖抑制作用。
PI3K
PI3K是细胞内重要的信号转导分子，根据PI3K的P110亚基结构特点和底物分子不同可将其分为三大类，其中第Ⅰ类PI3K功能最为重要。
PI3K主要由催化亚基P110和调节亚基P85组成。PI3K可被生长因子、细胞因子、激素等细胞外信号刺激激活。PI3K激活可使膜磷酸肌醇磷酸化，催化肌醇环上3位羟基生成3,4二磷酸磷脂酰肌醇(phosphatidylinositol-3,4-biphosphate，PI-3,4P2)及3,4,5三磷酸磷脂酰肌醇(phosphatidylinositol 3,4,5-trisphosphate，PI-3,4,5P3)，它们均可作为第二信使在细胞中传递信号，介导PI3K的多种细胞功能，如这些脂质产物可通过与Akt的PH(pleckstrin homology)区结合来激活Akt。肿瘤抑制基因pten(phosphatase and tensinhomologdelet-edonchromosometen)表达产物可诱导3磷酸肌醇去磷酸化，从而可对PI3K途径进行负调节。
Akt
Akt是一种Ser/Thr蛋白激酶。在磷脂酰肌醇依赖的蛋白激酶(phosphoinositide-dependent protein kinase，PDK)的协同作用下，PI-3,4P2和PI-3,4,5P3可与Akt结合，导致Akt从胞浆转位到质膜，并促进Akt的Ser473和Thr308位点磷酸化。
Ser473或/和Thr308位点的磷酸化是Akt激活的必要条件，而Akt激活是其发挥促细胞生存功能的重要前提。激活的Akt主要通过促进Bad(Bcl-2家族促凋亡成员之一)、mTOR、Caspase 3、糖原合酶激酶-3(glycogen synthasekinase-3,GSK-3)等下游底物磷酸化而发挥广泛的生物学效应，包括抗凋亡、促细胞生存等功能。
mTOR
mTOR也被称为FRAP(FKBP-rapamycin-associated protein)，属于磷酸肌醇激酶3相关激酶(PIKKs)家族的一员，是PI3K/Akt的下游底物，可通过改变 翻译调节因子4EBP1(真核细胞启动因子4E结合蛋白)和p70S6k的磷酸化状态启动翻译过程。
去磷酸化状态的4EBP1能与翻译起始因子eIF-4E结合，从而使后者丧失活性，而磷酸化的4EBP1可解离释放eIF-4E，从而使后者结合于mRNA 5'端起始点启动翻译过程。p70S6k磷酸化可通过促使40S核糖体蛋白S6磷酸化而启动翻译。PI3K/Akt/mTOR信号通路在正常和肿瘤细胞的增殖和生存中具有重要作用，以抑制mTOR为主要作用原理的抗肿瘤药物已经进入临床开发的早期阶段。
细胞周期调控
本发明提供了一种调控细胞周期剂的方法，所述方法包括，对对象细胞施用有效量的式I化合物或包括式I化合物的药物组合物。
较佳地，所述的式I化合物可以用于阻滞细胞周期，如用于诱导细胞产生G1周期阻滞，从而调控细胞的细胞周期，或改变细胞分裂的进程。
所述的式I化合物或其药物组合物可以选择性地调控部分细胞的细胞周期，而对于其他非敏感性细胞不起诱导周期阻滞的作用。在本发明的一个优选例中，所述的式I化合物或其药物组合物用于调控肿瘤细胞的细胞周期。
在另一优选例中，所述肿瘤细胞是选自下组的肿瘤细胞：Hela、H4、FHCC98、SMMC7721、BEL7404、A498、SW1116、MDA-MB-231、MDA-MB-468、DU145、U87-MG、Wi38、H1299细胞，或其组合；较佳地选自下组：Hela、H4、FHCC98、SMMC7721细胞，或其组合。
所述的式I化合物可以在较低浓度下，较佳地在≤1mM/L的浓度下作用于细胞并诱导或调控细胞的细胞周期，产生周期阻滞。在另一优选例中，所述式I化合物的有效浓度为0.01nM/L-1mM/L，较佳地为0.1nM/L-500μM/L，最佳地为1nM/L-100μM/L。
所述式I化合物的作用时间没有特别限制，如可以为2h～30天。较佳地，所述式I化合物的作用时间根据作用环境和作用对象不同而有所区别。在本发明的一个优选例中，所述式I化合物的作用时间为2～96h。在另一优选例中，所述式I化合物的作用时间为1～30天。
细胞凋亡诱导
本发明提供了一种诱导细胞凋亡或抑制细胞生长的方法，所述方法包括，对对象细胞施用有效量的式I化合物或包括式I化合物的药物组合物。
所述的式I化合物或其药物组合物可以选择性地诱导部分细胞凋亡或抑制部分细胞生长，而对于其他非敏感性细胞的生长不产生抑制作用。
在本发明的一个优选例中，所述的式I化合物或其药物组合物用于调控肿瘤细胞的细胞周期。较佳地，所述肿瘤细胞是选自下组的肿瘤细胞：Hela、H4、FHCC98、SMMC7721、BEL7404、A498、SW1116、MDA-MB-231、MDA-MB-468、DU145、U87-MG、Wi38、H1299细胞，或其组合；最优选的对象肿瘤细胞选自下组：Hela、H4、FHCC98、SMMC7721细胞，或其组合。
所述的式I化合物可以在较低浓度下，较佳地在≤100mM/L的浓度下作用于对象细胞并诱导或调控细胞的细胞周期，产生周期阻滞。
在另一优选例中，所述式I化合物的有效浓度为0.01nM/L-1mM/L，较佳地为0.1nM/L-500μM/L，最佳地为1nM/L-100μM/L。
所述式I化合物的作用时间没有特别限制，如可以为2h～30天。较佳地，所述式I化合物的作用时间根据作用环境和作用对象不同而有所区别。在本发明的一个优选例中，所述式I化合物的作用时间为2～96h。在另一优选例中，所述式I化合物的作用时间为1～30天。
抗肿瘤药物
本发明提供了一种诱导细胞凋亡或抑制细胞生长的方法，所述方法包括，对对象细胞施用有效量的式I化合物或包括式I化合物的药物组合物。
在本发明的一个优选例中，所述的药物组合物包括：(a)治疗有效量的式I化合物；和(b)药学上可接受的载体。
在另一优选例中，所述的肿瘤包括：宫颈癌、神经胶质瘤、肝癌。
在另一优选例中，所述式I化合物的有效浓度为0.01nM/L-1mM/L，较佳地为0.1nM/L-500μM/L，最佳地为1nM/L-100μM/L。
本发明还提供了一种抗肿瘤药物或药物组合物的制备方法，所述方法包括：将治疗有效量的式I化合物与药学上可接受的载体混合，从而形成药物组合物。
在另一优选例中，所述药物组合物还包括选自下组的组分：肿瘤抑制剂、肿瘤凋亡诱导剂，或其组合。
对被治疗对象施用治疗有效量的式I化合物或包括式I化合物的药物组合物，可以治疗或抑制肿瘤。
本发明的主要优点在于：
(1)首次揭示了甲基丙烯酰基苯并咪唑酮衍生物(FD系列)能够显著抑制抑制肿瘤细胞生长；
(2)首次揭示了甲基丙烯酰基苯并咪唑酮衍生物能够下调PI3K/Akt通路的活性；
(3)首次揭示了甲基丙烯酰基苯并咪唑酮衍生物能够降低肿瘤细胞的Akt活化水平；
(4)首次揭示了甲基丙烯酰基苯并咪唑酮衍生物能够显著抑制Akt的S473位点磷酸化的作用。
(5)首次揭示了甲基丙烯酰基苯并咪唑酮衍生物能够显著抑制mTOR激酶活性。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本文实施例中所用试剂及其它材料均可从市售渠道获得，其中生物试剂和材料如无特殊说明均购自上海研晶生物科技有限公司。
实施例1甲基丙烯酰基苯并咪唑酮衍生物对Akt磷酸化的抑制作用
酶联免疫吸附实验(ELISA)
采用双抗体夹心法建立ELISA筛选方法：将A549细胞(一种肺癌细胞，购自中国科学院上海生命科学研究院细胞资源中心)以一定浓度接种于12孔板中，37℃二氧化碳培养箱培养过夜，使细胞贴壁，同时，将Akt抗体以特定浓度接种到高吸附的酶标板中，置于4℃的冰箱中；无菌PBS清洗12孔板三次，加入无血清的F12，培养箱培养24小时；在12孔板中加入MK2206、Triciribine和FD系列药物，作用1小时，每孔中加入100ng的IGF-1刺激10分钟；预冷的PBS清洗12孔板三次，每孔加入100μl RIPA裂解液，冰上裂解20分钟，将裂解液收集并加入相应的酶标板(购自圣克鲁斯生物技术公司)孔中，冰上振摇；在酶标板中加入p-Akt(S473)抗体(购自赛信通生物试剂有限公司)，振摇一段时间后，加入抗兔抗体(购自赛信通生物试剂有限公司)，TMB显色后以2M的硫酸终止反应，酶标仪上测定450nm处的吸收值，抑制率＝(OD样品-OD-IGF)/(ODIGF-OD-IGF),其中OD样品是指待测样品孔的吸收值，ODIGF是指只加入IGF孔的吸收值，OD-IGF是指未加入IGF孔的吸收值。结果如表1所示。阳性对照为MK2206和Triciribine(曲西立宾),在细胞水平对Akt磷酸化的抑制率分别为92％和46％，本发明所涉及的化合物中FD1和FD3对Akt磷酸化的抑制率分别为85％和91％,优于阳性对照Triciribine,与阳性对照MK2206相当，FD2，FD4，FD12，FD13与Triciribine作用相当。
实验结果证明FD系列化合物(甲基丙烯酰基苯并咪唑酮衍生物)，对Akt的磷酸化具有显著的抑制作用，其中部分化合物的抑制效果与处于临床试验阶段的阳性化合物相当。
表1
Figure PCTCN2015074896-appb-000020
Figure PCTCN2015074896-appb-000021
蛋白质免疫印迹
A549细胞以2×105细胞/孔的密度种入12孔板，贴壁过夜，弃去培养液并用生理盐水清洗三次，加入新鲜的雾血清培养基饥饿24小时，加入待测化合物作用1h，然后加入IGF-Ⅰ刺激10min,加入细胞裂解液与冰上裂解半小时，收集细胞裂解产物，20000×g 4℃离心10min后取上清，用MicroBCA蛋白定量试剂盒进行蛋白定量，并调整蛋白浓度使均一。利用4×SDS上样缓冲液[200mM Tris.Cl(pH 6.8)、400mM DTT、8％SDS、0.4％溴酚蓝、40％甘油]混合后煮沸15min。室温自然冷却，将等量样品上样于聚丙烯酰胺凝胶，在Tris-甘氨酸电泳缓冲液(25mM Tris、250mM甘氨酸、0.1％SDS)中以80-120V电泳约2h。用半干法将蛋白从凝胶转移至硝酸纤维素滤膜。转移条件为：转移缓冲液(39mM甘氨酸、48mM Tris碱、0.037％SDS、20％甲醇)；转移电流25mA；转移时间2h。转移结束，用丽春红(Ponceau S)染色确定转移情况和蛋白条带在硝酸纤维素滤膜上的位置，标记后用含5％脱脂奶粉的封闭液[5％脱脂奶粉，20mM Tris-HCl(pH 7.2-7.4)、150mM NaCl、0.1％(v/v)Tween20]于摇床室温封闭2h。加入特异性一抗于4℃杂交过夜。用PBST洗液[20mM Tris-HCl(pH  7.2-7.4)、150mM NaCl、0.1％(v/v)Tween20]室温洗3次，15min/次。加入辣根过氧化物酶标记的二抗，室温置于摇床上平缓摇动2h。PBST洗液洗三次后，用SuperSignal West Dura Chemiluminescent Substrate(Pierce Inc,Rochford,IL)发色后暴光、显影、定影。结果如图1所示。图中共12个泳道，条带颜色深浅和大小代表相应蛋白浓度的高低，Akt的12条泳道中显示出条带颜色深浅和大小基本相同，说明各泳道中总Akt量相同；而p-Akt(S473)的12条泳道中条带的颜色深浅和条带大小差异很大，说明各个条带中的p-Akt(S473)的量有差异；IGF可以刺激信号通路中Akt磷酸化水平的快速升高，对照1中在细胞培养液中仅加入100ng/ml的IGF，Akt磷酸化水平显著升高，而对照2中细胞培养液中未加入IGF，Akt磷酸化水平较低；MK2206是默克制药开发的一种Akt磷酸化抑制剂，目前处于临床二期试验阶段，对多种肿瘤显示出良好的治疗效果，图1中所示，MK2206对应的条带与对照1的条带相比，显著变小，说明MK2206显著抑制IGF刺激后的Akt磷酸化水平的升高；FD1和FD3显示出与MK2206相当的作用效果。实验结果证明FD系列化合物(甲基丙烯酰基苯并咪唑酮衍生物)，对Akt的S473位点的磷酸化具有显著的抑制作用。
实施例2甲基丙烯酰基苯并咪唑酮衍生物对肿瘤细胞的抑制作用
磺酰罗丹明B法
在96孔板中种入一定数量的细胞，贴壁过夜，加入待测化合物作用72h后，弃去培养液，加入100μL/孔三氯乙酸(TCA)于4℃固定2h后弃去固定液，用蒸馏水轻柔洗5次，室温干燥。加入100μL/孔4mg/mL SRB染色液，室温染色15min,弃去染色液并用1％冰醋酸(HAC)洗5次，室温干燥。加入150μL/孔Tris base buffer(10mM)，室温放置15min,酶标仪540nm波长下测定OD值。抑制率(％)＝(OD值对照孔-OD值给药孔)/OD值对照孔×100％。
结果如表2所示，MK2206和FD系列化合物作用浓度为10μM。表中所示PC-3为前列腺癌细胞株购自中国科学院上海生命科学研究院细胞资源中心，A549为肺癌细胞株购自中国科学院上海生命科学研究院细胞资源中心，SGC-7901为胃癌细胞株购自中国科学院上海生命科学研究院细胞资源中心，结果表明FD1能够抑制肿瘤细胞株增殖，对PC-3肿瘤细胞(PTEN缺失)的增殖抑制更为显著，提示FD1为代表的FD系列化合物对PTEN缺失的肿瘤类型有更好的治疗效果。
表2
Figure PCTCN2015074896-appb-000022
Figure PCTCN2015074896-appb-000023
实施例3
在本实施例中，采用实施例1中相同的方法，测定下列化合物对Akt的磷酸化的抑制作用。
公开于CN201110326737中表1-表4中以下的化合物：
(1)化合物IA-01至IA-195中的IA-01，IA-162；
(2)化合物Ia-01至Ia-195中的Ia-05,Ia-134；
(3)化合物IB-01至IB-195中的IB-07，IB-075；
(4)化合物Ib-01至Ib-195中的Ib-07,Ib-09；
(5)化合物IC-01至IC-195中的IC-137；
(6)化合物Ic-01至Ic-195中的Ic-06,Ic-135；
(7)化合物ID-01至ID-195中的ID-014；
(8)化合物Id-01至Id-195中的Id-01；
(9)化合物IIA-01至IIA-45中的IIA-41；
(10)化合物IIa-01至IIa-45中的IIa-13,IIa-39；
(11)化合物IIB-01至IIB-45中的IIB-08，IIB-32；
(12)化合物IIb-01至IIb-45中的IIb-46；
(13)化合物IIC-01至IIC-45中的IIC-26；
(14)化合物IIc-01至IIc-45中的IIc-16；
(15)化合物IID-01至IID-45中的IID-32；
(16)化合物IId-01至IId-45中的IId-17,IId-36；
(17)化合物IIIA-01至IIIA-60中的IIIA-03，IIIA-37；
(18)化合物IIIa-01至IIIa-60中的IIIa-01；
(19)化合物IIIB-01至IIIB-60中的IIIB-60；
(20)化合物IIIb-01至IIIb-60中的IIIb-60；
(21)化合物IIIC-01至IIIC-60中的IIIC-14；
(22)化合物IIIc-01至IIIc-60中的IIIc-27,IIIc-59；
(23)化合物IIID-01至IIID-60中的IIID-29；
(24)化合物IIId-01至IIId-60中的IIId-27；
(25)化合物IVA-01至IVA-51中的IVA-51；
(26)化合物IVa-01至IVa-51中的IVa-43；
(27)化合物IVB-01至IVB-51中的IVB-38，IVB-45；
(28)化合物IVb-01至IVb-51中的IVb-28；
(29)化合物IVC-01至IVC-51中的IVC-05，IVC-27；
(30)化合物IVc-01至IVc-51中的IVc-37；
(31)化合物IVD-01至IVD-51中的IVD-13，IVD-46；
(32)化合物IVd-01至IVd-51中的IVd-47。
结果表明，所有测试的化合物都对Akt的磷酸化具有显著的抑制作用，当其浓度为10μM时，对Akt的磷酸化抑制率为3％-91％，大多数(3/4以上)的抑制率为30％－60％。
实施例4肿瘤相关激酶的抑制活性测试
本发明测试了FD162化合物对114个肿瘤相关激酶的抑制活性，发现FD162(浓度10uM)能够选择性的抑制mTOR的活性。此实验委托Eurofins Pharma Discovery Services UK Limited完成并出具报告。实验结果如下表所示。
Figure PCTCN2015074896-appb-000024
Figure PCTCN2015074896-appb-000025
实施例5肿瘤细胞株的增殖抑制活性
本发明测试了代表性化合物FD162对20多株肿瘤细胞株的增殖抑制活性，实验方法同实施例2。结果如图2所示，表明FD162能够普遍的抑制肿瘤细胞的增殖，其中FD162对Hela细胞增殖的半数抑制浓度为3.67微摩尔。
实施例6信号通路抑制实验
采用实施例1中的方法，在细胞水平测试了不同浓度FD162对信号通路的影响，如图4所示，表明FD162能够浓度依赖的下调PI3K/Akt/mTOR信号通路。RAD为已上市的mTOR抑制剂抗肿瘤药物依维莫司，浓度为10uM,Rapa为雷帕霉素，MK为处于临床试验阶段的Akt抑制剂抗肿瘤药物MK2206。
实施例7动物活性实验
动物活性实验采用的方法为Hela细胞裸鼠皮下移植瘤。
具体实验方法如下：
取生长旺盛期的瘤组织剪切成1.5mm3左右，在无菌条件下，接种于裸小鼠右侧腋窝皮下。裸小鼠皮下移植瘤用游标卡尺测量移植瘤直径，待肿瘤生长至100-200mm3后将动物随机分组。FD162按照20mg/kg,10mg/kg和5mg/kg分成三组，每组9只；雷帕霉素按10mg/kg组；模型组给生理盐水，每天腹腔注射给药一次，给药周期为12天。整个实验过程中，每周2次测量移植瘤直径，同时称量小鼠体重。肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×a×b2，其中a、b分别表示长、宽。根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝Vt/V0。其中V0为分笼给药时(即d0)测量所得肿瘤体积，Vt为每一次测量时的肿瘤体积。抗肿瘤活性的评价指 标为1)相对肿瘤增殖率T/C(％)，计算公式如下：T/C(％)＝(TRTV/CRTV)×100％，TRTV：治疗组RTV；CRTV：阴性对照组RTV；2)肿瘤体积增长抑制率GI％，计算公式如下：GI％＝[1-(TVt-TV0)/(CVt-CT0)]×100％，TVt为治疗组每次测量的瘤体积；TV0为治疗组分笼给药时所得瘤体积；CVt为对照组每次测量的瘤体积；CV0为对照组分笼给药时所得瘤体积；3)瘤重抑制率，计算公式如下：瘤重抑制率％＝(Wc-WT)/Wc×100％，Wc：对照组瘤重，WT：治疗组瘤重。
实验结果表明，本发明的化合物FD162在中剂量组(10mg/kg)的情况下对肿瘤的抑制效果最好，优于阳性对照组，给药12天后中剂量组(10mg/kg)肿瘤抑制率达到了50％以上。肿瘤抑制效果如图5所示。
实施例8FD162化合物的制备
本发明公开的FD162化合物可采用如下合成路线制备。
Figure PCTCN2015074896-appb-000026
具体操作步骤如下：
中间体62的合成。在装有分水器的三口反应瓶中，加入187.0g(1mol)的5-溴-2,3-二氨基吡啶(60)和450mL二甲苯，5mL氢氧化钾乙醇溶液(其中氢氧化钾重量为1.0g)，加热搅拌回流后，缓慢滴加156.0g(1.2mol)乙酰乙酸乙酯(2)和160mL二甲苯混合液，滴完后，搅拌回流共沸脱水至无水珠出现为止，再继续搅拌回流2h。反应毕，冷却，析出结晶，过滤、干燥，得白色粒状结晶235.5g(产率93.1％)，即为6-溴-1-异丙烯基-1H-咪唑[4,5-并]吡啶-2(3H)-酮(62)，熔点130～132℃，质谱(ESI，正离子模式)[M+H]+m/z 254。
FD162的合成。在三口反应瓶中加入2.53g(0.01mol)6-溴-1-异丙烯基-1H-咪唑[4,5-并]吡啶-2(3H)-酮(62)和50mL经干燥处理的二氯甲烷，搅拌至完全溶解，再加入2mL三乙胺。冰浴下滴加0.86g(0.011moL)乙酰氯，加完后恢复至室温下搅拌5～8h。反应结束后，加入50mL饱和碳酸氢钠溶液，继续搅拌15min，转入分液漏斗中弃去水相，有机相经水洗、饱和氯化钠溶液洗涤，再经无水硫酸钠干燥后减压脱去溶剂，浓缩液经硅胶柱层析纯化(洗脱剂为石油醚/乙酸乙酯＝6/1)，即得FD1622.72g(产率92.2％)，熔点136～138℃。质谱(ESI，正离子模式)[M+H]+m/z 296。1H NMR(CDCl3),δ2.23(s,3H)，2.76(s,3H)，5.35(s,1H),5.51(s,1H),8.23(d,J＝2.0Hz,1H),8.53(d,J＝2.0Hz,1H)；13C NMR(CDCl3),δ20.2,25.6,114.1,116.2,121.5,125.2,135.2,141.9,144.4,149.9,170.2。
讨论
本发明所涉及的甲基丙烯酰基苯并咪唑酮衍生物能够下调P13K/Akt信号通路中Akt磷酸化水平，作用效果与新型小分子靶向药物MK2206相当；而细胞水平的研究发现，以FD1为代表的甲基丙烯酰基苯并咪唑酮衍生物对肿瘤细胞具有良好的增殖抑制效果，与MK2206有所不同的是，FD1对PTEN缺失型的细胞作用效果更好。
本发明的甲基丙烯酰基苯并咪唑酮衍生物对mTOR激酶具有及其显著的抑制效果，能够开发为用于肿瘤治疗的mTOR激酶抑制剂。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种甲基丙烯酰基苯并咪唑酮衍生物，其光学异构体，或其药学上可接受的盐或前药的用途，其特征在于，用于制备：
    (a)用于下调PI3K/Akt通路的活性的药物组合物或试剂；
    (b)用于治疗或抑制肿瘤和/或抑制肿瘤细胞生长的药物组合物或试剂；
    (c)用于阻滞细胞周期的药物组合物或试剂；和/或
    (d)用于制备抑制mTOR激酶活性的药物组合物或试剂。
  2. 如权利要求1所述的用途，其特征在于，所述甲基丙烯酰基苯并咪唑酮衍生物具有式I结构；
    Figure PCTCN2015074896-appb-100001
    其中，
    R代表：
    氢；C1～C8的烷基；或任选被氰基，卤素，苯环，C1～C4烷氧基或C1～C4烷硫基取代的C1～C8烷基，C3～C8链烯基或C3～C8炔基；或任选被氰基，卤素，C1～C4烷氧基或C1～C4烷硫基取代的C3～C8链烯基或C3～C8炔基；苯基或由选自下列的1～3个取代基任选取代的苯基：卤素，C1～C8烷基，C3～C8环烷基，羟基，C1～C4烷氧基，C1～C4卤代烷基，C1～C4卤代烷氧基，C1～C4卤代烷硫基，C1～C4卤代烷基磺酰基，羧基，硝基，氰基，苯基，苯氧基，苯甲酰基；
    Y代表：
    氢；或选自下列的1～4个取代基：卤素，硝基，C1～C8烷基，C3～C8环烷基，C1～C4卤代烷基；
    其中，当Y代表氢时，R不为异丙烯基；
    或者Y代表：
    Figure PCTCN2015074896-appb-100002
    其中，Z1代表：羟基，C1～C8烷氧基，C3～C8烯氧基或C3～C8炔氧基，氨基，或者被1个或2个C1～C8烷基取代的胺基；
    或者Y代表：
    -O(S)-Z2
    其中，Z2代表：氢，C1～C4烷基，C3～C8链烯基或C3～C8炔基，C1～C4卤代烷基，C1～C8脂肪酰基，C1～C8卤代脂肪酰基，氨基甲酰基，N-甲基胺基甲酰基，N-乙基胺基甲酰基，N,N-二甲基胺基甲酰基，N,N-二乙基胺基甲酰基，苯基，苯甲酰基，苯乙酰基，苯磺酰基或由选自下列的1～3个取代基任选取代的苯基，苯甲酰基或苯磺酰基：卤素，C1～C8烷基，C3～C8环烷基，羟基，C1～C4烷氧基，C1～C4卤代烷基，C1～C4卤代烷氧基，C1～C4卤代烷硫基，C1～C4卤代烷基磺酰基，羧基，硝基，氰基；
    或者Y代表：
    Figure PCTCN2015074896-appb-100003
    其中，Z3代表氢，C1～C8烷基，C3～C8环烷基，C3～C8链烯基，C3～C8链炔基，C1～C4卤代烷基，C1～C8酰基，或带有1～3个选自氮、氧和硫的杂原子的的5元或6元杂环基，苯基，苯甲酰基，苯乙酰基，苯磺酰基或由选自下列的1～3个取代基任选取代的苯基，苯甲酰基或苯磺酰基：卤素，C1～C8烷基，C3～C8环烷基，羟基，C1～C4烷氧基，C1～C4卤代烷基，C1～C4卤代烷氧基，C1～C4卤代烷硫基，C1～C4卤代烷基磺酰基，羧基，硝基，氰基；Z4代表氢，C1～C8烷基，C3～C8环烷基，C3～C8链烯基，C3～C8链炔基，C1～C4卤代烷基，或Z3和Z4与它们所连接的氮原子一起形成5～7元杂环基，除了氮原子以外，该杂环基可以含有一个或两个以上选自氮和氧的杂原子。
  3. 如权利要求1所述的用途，其特征在于，所述的药物组合物或试剂用于降低Akt活化水平；和/或，所述的药物组合物或试剂用于抑制Akt的磷酸化。
  4. 如权利要求1所述的用途，其特征在于，所述的肿瘤或肿瘤细胞选自：肝癌、宫颈癌、神经胶质瘤、结肠癌、肾癌、乳腺癌、肺癌、前列腺癌、胰腺癌、皮肤癌、鼻咽癌、食道癌、胃癌、卵巢癌和黑色素瘤；优选地，所述肿瘤或肿瘤细胞为PTEN突变(PTEN表达水平下降)或缺失的肿瘤或肿瘤细胞。
  5. 如权利要求1所述的用途，其特征在于，所述甲基丙烯酰基苯并咪唑酮衍生物具有式II所示结构，
    Figure PCTCN2015074896-appb-100004
    式II中，
    A选自：C、或N；
    R1、R5分别独立地选自：氢、卤素、C1～C10烷基、C2～C8烯基、C2～C8炔基、CN、NO2；
    R2、R3分别独立地选自：氢、卤素、C1～C10烷基、C2～C8烯基、C2～C8炔基、C3～C10环烷基、杂环基、芳基、杂芳基、C1～C10醛基、C2～C10酰基、C2～C10酯基、CN、NO2；
    R4选自：O、S、卤素；
    Figure PCTCN2015074896-appb-100005
    表示单键或双键；
    所述烷基、烯基、炔基、环烷基、杂环基、芳基、杂芳基、醛基、酰基、酯基为取代的或非取代的。
  6. 一种药物组合物，其特征在于，所述的药物组合物包括：(a)式I或式II所示的甲基丙烯酰基苯并咪唑酮衍生物，其光学异构体，或其药学上可接受的盐或前药；和(b)药学上可接受的载体，
    Figure PCTCN2015074896-appb-100006
    式中，各基团的定义如上所述。
  7. 一种体外非治疗性的下调PI3K/Akt通路的活性和/或调节细胞周期的方法，其特征在于，所述方法包括步骤：在式I或式II所示的甲基丙烯酰基苯并咪唑酮衍生物，其光学异构体，或其药学上可接受的盐或前药存在下，培养所述细胞，从而在所述细胞中下调PI3K/Akt通路的活性和/或调节细胞周期。
  8. 一种体外非治疗性抑制细胞生长或诱导细胞凋亡的方法，其特征在于，所述方法包括步骤：在式I或式II所示的甲基丙烯酰基苯并咪唑酮衍生物，其光学异构体，或其药学上可接受的盐或前药存在下，培养所述细胞。
  9. 一种式I所示的甲基丙烯酰基苯并咪唑酮衍生物或其药学上可接受的盐的用途，其特征在于，用于制备组合物，所述组合物用于选自下组的一种或多种应用：
    (i)抑制肿瘤细胞生长；
    (ii)诱导肿瘤细胞凋亡；
    (iii)干扰肿瘤细胞增殖；
    (iv)调控肿瘤细胞的细胞周期；
    (v)抑制肿瘤细胞的转移；
    (vi)下调PI3K/Akt通路的活性；
    (vii)降低Akt活化水平；
    (viii)抑制Akt的磷酸化；
    (ix)抑制Akt S473位点的磷酸化；
    (x)治疗或预防肿瘤；
    (xi)降低mTOR活化水平；
    (xii)抑制mTOR的磷酸化；和
    (xii)下调PI3K/Akt/mTOR信号通路的活性。
  10. 一种用于治疗或抑制肿瘤的药物组合物，所述的药物组合物含有(a)药学上可接受的载体和(b)式I或式II化合物，其光学异构体，或其药学上可接受的盐或前药，
    Figure PCTCN2015074896-appb-100007
    式中，各基团的定义如上所述。
  11. 一种药盒，其特征在于，所述药盒包含：
    一容器，以及位于所述容器内的式I所示的甲基丙烯酰基苯并咪唑酮衍生 物，其光学异构体，或其药学上可接受的盐或前药，以及药学上可接受的载体；
    一说明书，所述说明书记载了将所述甲基丙烯酰基苯并咪唑酮衍生物用于选自下组的一种或多种应用：
    (i)抑制肿瘤细胞生长；
    (ii)诱导肿瘤细胞凋亡；
    (iii)干扰肿瘤细胞增殖；
    (iv)调控肿瘤细胞的细胞周期；
    (v)抑制肿瘤细胞的转移；
    (vi)下调PI3K/Akt通路的活性；
    (vii)降低Akt活化水平；
    (viii)抑制Akt的磷酸化；
    (ix)抑制Akt S473位点的磷酸化；
    (x)治疗或预防肿瘤；
    (xi)降低mTOR活化水平；
    (xii)抑制mTOR的磷酸化；和
    (xii)下调PI3K/Akt/mTOR信号通路的活性。
  12. 一种化合物，所述化合物具有式II所示结构，
    Figure PCTCN2015074896-appb-100008
    式II中，A选自：C、或N；
    R1、R5分别独立地选自：氢、卤素、C1～C10烷基、C2～C8烯基、C2～C8炔基、CN、NO2；
    R2、R3分别独立地选自：氢、卤素、C1～C10烷基、C2～C8烯基、C2～C8炔基、C3～C10环烷基、杂环基、芳基、杂芳基、C1～C10醛基、C2～C10酰基、C2～C10酯基、CN、NO2；
    R4选自：O、S、卤素、；
    Figure PCTCN2015074896-appb-100009
    表示单键或双键；所述烷基、烯基、炔基、环烷基、杂环基、芳基、杂芳基、醛基、酰基、酯基为取代的或非取代的。
  13. 如权利要求12所述化合物，其中R1选自：氢、卤素或三氟甲基。
  14. 如权利要求12所述化合物，其中A为N。
  15. 如权利要求12所述化合物，其中所述化合物为
    Figure PCTCN2015074896-appb-100010
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